Phosphodiesterase 10

ABSTRACT

The present invention provides novel human PDE10 polypeptides, polynucleotides encoding the polypeptides, expression constructs comprising the polynucleotides, host cells transformed with the expression constructs; methods for producing PDE10 polypeptides; antisense polynucleotides; and antibodies specifically immunoreactive with the PDE10 polypeptides. The invention further provides methods to identify binding partners of PDE 10, and more particularly, binding partners that modulate PDE10 enzyme activity.

[0001] This application claims priority of U.S. Provisional ApplicationNo. 60/075,508, filed Feb. 23, 1998.

FIELD OF THE INVENTION

[0002] The present invention relates generally to a novelphosphodiesterase (PDE) designated PDE10. Depending on nomenclatureused, PDE10 is also referred to as PDE9.

BACKGROUND OF THE INVETION

[0003] Phosphodiesterases (PDEs) hydrolyze 3′,5′ cyclic nucleotides totheir respective nucleoside 5′ monophosphates. The cyclic nucleotidescAMP and cGMP are synthesized by adenylyl and guanylyl cyclases,respectively, and serve as second messengers in a number of cellularsignaling pathways. The duration and strength of the second messengersignal is a function of the rate of synthesis and the rate of hydrolysisof the cyclic nucleotide.

[0004] Multiple families of PDEs have been identified. The nomenclaturesystem includes first a number that indicates the PDE family. To date,nine families (PDE1-9) are known which are classified by: (i) primarystructure; (ii) substrate preference; (iii) response to differentmodulators; (iv) sensitivity to specific inhibitors; and (v) modes ofregulation [Loughney and Ferguson, in Phosphodiesterase Inhibitors,Schudt, et al (Eds.), Academic Press: New York, N.Y. (1996) pp. 1-19].The number indicating the family is followed by a capital letter,indicating a distinct gene, and the capital letter followed by a secondnumber, indicating a specific splice variant or a specific transcriptthat utilizes a unique transcription initiation site.

[0005] The amino acid sequences of all mammalian PDEs identified to dateinclude a highly conserved region of approximately 270 amino acidslocated in the carboxy terminal half of the protein [Charbonneau, etal., Proc. Natl. Acad. Sci. (USA) 83:9308-9312 (1986)]. The conserveddomain includes the catalytic site for cAMP and/or cGMP hydrolysis andtwo putative zinc binding sites as well as family specific determinants[Beavo, Physiol. Rev. 75:725-748 (1995); Francis, et al., J. Biol. Chem.269:22477-22480 (1994)]. The amino terminal regions of the various PDEsare highly variable and include other family specific determinants suchas: (i) calmodulin binding sites (PDE1); (ii) non-catalytic cyclic GMPbinding sites (PDE2, PDE5, PDE6); (iii) membrane targeting sites (PDE4);(iv) hydrophobic membrane association sites (PDE3); and (v)phosphorylation sites for either the calmodulin-dependent kinase II(PDE1), the cAMP-dependent kinase (PDE1, PDE3, PDE4), or the cGMPdependent kinase (PDE5) [Beavo, Physiol. Rev. 75:725-748 (1995);Manganiello, et al., Arch. Biochem. Acta 322:-13 (1995); Conti, et al,Physiol. Rev. 75:723-748 (1995)].

[0006] Members of the PDE1 family are activated by calcium-calmodulin.Three genes have been identified; PDE1A and PDE1B preferentiallyhydrolyze cGMP while PDE1C has been shown to exhibit a high affinity forboth cAMP and cGMP. The PDE2 family is characterized as beingspecifically stimulated by cGMP [Loughney and Ferguson, supra]. Only onegene has been identified, PDE2A, the enzyme product of which isspecifically inhibited by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA).Enzymes in the PDE3 family are specifically inhibited by cGMP. Two genesare known, PDE3A and PDE3B, both having high affinity for both cAMP andcGMP, although the V_(max) for cGMP hydrolysis is low enough that cGMPfunctions as a competitive inhibitor for cAMP hydrolysis. PDE3 enzymesare specifically inhibited by milrinone and enoximone [Loughney andFerguson, supra]. The PDE4 family effects cAMP hydrolysis and includesfour genes, PDE4A, PDE4B, PDE4C, and PDE4D, each having multiple splicevariants. Members of this family are specifically inhibited by theanti-depressant drug rolipram. Members of PDE5 family bind cGMP atnon-catalytic sites and preferentially hydrolyze cGMP. Only one gene,PDE5A, has been identified. The photoreceptor PDE6 enzymes specificallyhydrolyze cGMP [Loughney and Ferguson, supra]. Genes include PDE6A andPDE6B (the protein products of which dimerize and bind two copies of asmaller γ inhibitory subunit to form rod PDE), in addition to PDE6Cwhich associates with three smaller proteins to form cone PDE. The PDE7family effects cAMP hydrolysis but, in contrast to the PDE4 family, isnot inhibited by rolipram [Loughney and Ferguson, supra]. Only one gene,PDE7A, has been identified. The PDE8 family has been shown to hydrolyzeboth cAMP and cGMP and is insensitive to inhibitors specific for PDEs1-5. Depending on nomenclature used, PDE8 is also referred to as PDE10,but is distinct from PDE10 described herein. The PDE9 familypreferentially hydrolyzes cAMP and is not sensitive to inhibition byrolipram, a PDE4-specific inhibitor, or isobutyl methyl xanthine (IBMX),a non-specific PDE inhibitor. Depending on nomenclature used, PDE9 isalso referred to as PDE8, but is distinct from PDE8 mentioned above. Todate, two genes have been identified in the PDE9 family.

[0007] Specific and non-specific inhibitors of the various PDE proteinfamilies have been shown to be effective in treating disordersattributable, in part, to aberrant levels of cAMP or cGMP. For example,the PDE4-specific inhibitor rolipram, mentioned above as ananti-depressant, inhibits lipopolysaccharide-induced expression of TNFαand has been effective in treating multiple sclerosis in an animalmodel. Other PDE4-specific inhibitors are being investigated for use asanti-inflammatory therapeutics, and efficacy in attenuating the lateasthmatic response to allergen challenge has been demonstrated[Harbinson, et al., Eur. Respir. J. 10:1008-1014 (1997)]. inhibitorsspecific for the PDE3 family have been approved for treatment ofcongestive heart failure. PDE5 inhibitors are currently being evaluatedfor treatment of penile erectile dysfunction [Boolell, et al., Int. J.Impotence Res. 8:47-50 (1996)]. Non-specific inhibitors, such astheophylline and pentoxifylline, are currently used in the treatment ofrespiratory and vascular disorders, respectively.

[0008] Given the importance of cAMP and cGMP in intracellular secondmessenger signaling, there thus exists an ongoing need in the art toidentify additional PDE species. Identification of heretofore unknownfamilies of PDEs, and genes and splice variants thereof, will provideadditional pharmacological approaches to treating conditions in whichcyclic nucleotide pathways are aberrant, as well as conditions in whichmodulation of intracellular cAMP and/or cGMP levels in certain celltypes is desirable. Identification of family-specific andenzyme-specific inhibitors will permit development of therapeutic andprophylactic agents which act on desired cell types expressing PDEsand/or particular metabolic pathways regulated by cyclic nucleotidemonophosphate steady-state concentrations.

SUMMARY OF THE INVENTION

[0009] In brief, the prevent invention provides purified and isolatedPDE10 polypeptides. Preferred polypeptides comprise the amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:18, SEQ ID NO: 20 and SEQ ID NO: 22.

[0010] The invention also provides polynucleotides encoding polypeptidesof the invention. A preferred polynucleotide comprises the sequence setforth in SEQ ID NO: 1. Polynucleotides of the invention includepolynucleotides encoding a human PDE10 polypeptide selected from thegroup consisting of a) the polynucleotide according to SEQ ID NO: 1, 18,20 or 22; b) a DNA which hybridizes under moderately stringentconditions to the non-coding strand of the polynucleotide of (a); and c)a DNA which would hybridize to the non-coding strand of thepolynucleotide of (a) but for the redundancy of the genetic code.Polynucleotides of the invention comprise any one of the polynucleotideset out in SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22, as well asfragments thereof. The invention provide polynucleotides which are DNAmolecules. DNA molecules include cDNA, genomic DNA, and wholly orpartially chemically synthesized DNA molecule. The invention alsoprovides antisense polynucleotides which specifically hybridizes withthe complement of a polynucleotide of the invention.

[0011] The invention also provides expression constructs comprising apolynucleotide of the invention, host cells transformed or transfectedwith an expression construct of the invention, and methods for producinga PDE10 polypeptide comprising the steps of: a) growing the host cell ofthe invention under conditions appropriate for expression of the PDE10polypeptide and b) isolating the PDE10 polypeptide from the host cell orthe medium of its growth.

[0012] The invention further provides antibodies specificallyimmunoreactive with a polypeptide of the invention. Preferably, theantibody is a monoclonal antibody. The invention also provideshybridomas which produces an antibody of the invention. Anti-idiotypeantibody specifically immunoreactive with the antibody of the inventionare also contemplated.

[0013] The invention also provides methods to identify a specificbinding partner compound of a PDE10 polypeptide comprising the steps of:a) contacting the PDE10 polypeptide with a compound under conditionswhich permit binding between the compound and the PDE10 polypeptide; b)detecting binding of the compound to the PDE10 polypeptide; and c)identifying the compound as a specific binding partner of the PDE10polypeptide. Preferably, methods of the invention identify specificbinding partners that modulate activity of the PDE10 polypeptide. In oneaspect, the methods identify compounds that inhibits activity ofthePDE10 polypeptide. In another aspect, the methods identify compoundsthat enhance activity of the PDE10 polypeptide.

[0014] The invention also provides methods to identify a specificbinding partner compound of the PDE10 polynucleotide of the inventioncomprising the steps of: a) contacting the PDE10 polynucleotide with acompound under conditions which permit binding between the compound andthe PDE10 polynucleotide; b) detecting binding of the compound to thePDE10 polynucleotide; and c) identifying the compound as a specificbinding partner of the PDE 10 polynucleotide. Preferably, the methodsidentify specific binding partner compounds that modulate expression ofa PDE10 polypeptide encoded by the PDE10 polynucleotide. In one aspect,method of the invention identify compounds that inhibit expression ofthe PDE10 polypeptide. In another aspect, methods of the inventionidentify compounds that enhance expression of the PDE10 polypeptide.

[0015] Binding partner compounds identified by methods of the inventionare also contemplated, as are compositions comprising the compound. Theinvention further comprehends use of binding partner compounds of theinvention in production of medicaments for the treatment of PDE10-related disorders.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides polypeptides and underlyingpolynucleotides for a novel PDE family designated PDE10. The PDE10family is distinguished from previously known PDE families in that itshows a lower degree of sequence homology than would be expected for amember of a known family of PDEs and it is not sensitive to inhibitorsthat are known to be specific for previously identified PDE families.Outside of the catalytic region of the protein, PDE10 shows littlehomology to other known PDEs. Even over the catalytic region, PDE10amino acid sequence identity is less than 40% when compared with thesame region in known PDEs. The invention includes both naturallyoccurring and non-naturally occurring PDE10 polynucleotides andpolypeptide products thereof Naturally occurring PDE10 products includedistinct gene and polypeptide species within the PDE10 family; thesespecies include those which are expressed within cells of the sameanimal as well as corresponding species homologs expressed in cells ofother animals. Within each PDE10 species, the invention further providessplice variants encoded by the same polynucleotide but which arise fromdistinct mRNA transcripts. Non-naturally occurring PDE10 productsinclude variants of the naturally occurring products such as analogs(i.e., wherein one or more amino acids are added, substituted, ordeleted) and those PDE10 products which include covalent modifications(i.e., fusion proteins, glycosylation variants, and the like).

[0017] The present invention provides novel purified and isolatedpolynucleotides (e.g., DNA sequences and RNA transcripts, both sense andcomplementary antisense strands, including splice variants thereof)encoding human PDE10 s. DNA sequences of the invention include genomicand cDNA sequences as well as wholly or partially chemically synthesizedDNA sequences. Genomic DNA of the invention comprises the protein codingregion for a polypeptide of the invention and includes allelic variantsof the preferred polynucleotide of the invention. Genomic DNA of theinvention is distinguishable from genomic DNAs encoding polypeptidesother than PDE10 in that it includes the PDE10 coding region as definedby PDE10 cDNA of the invention. The invention therefore providesstructural, physical, and functional characterization for genomic PDE 10DNA. Allelic variants are known in the art to be modified forms of awild type gene sequence, the modification resulting from recombinationduring chromosomal segregation or exposure to conditions which give riseto genetic mutation. Allelic variants, like wild type genes, areinherently naturally occurring sequences (as opposed to non-naturallyoccurring variants which arise from in vitro manipulation).“Synthesized,” as used herein and is understood in the art, refers topurely chemical, as opposed to enzymatic, methods for producingpolynucleotides. “Wholly” synthesized DNA sequences are thereforeproduced entirely by chemical means, and “partially” synthesized DNAsembrace those wherein only portions of the resulting DNA were producedby chemical means. A preferred DNA sequence encoding a human PDE10polypeptide is set out in SEQ ID NO: 1. The worker of skill in the artwill readily appreciate that the preferred DNA of the inventioncomprises a double stranded molecule, for example the molecule havingthe sequence set forth in SEQ ID NO: 1 along with the complementarymolecule (the “non-coding strand” or “complement”) having a sequencededucible from the sequence of SEQ ID NO: 1 according to Watson-Crickbase paring rules for DNA. Also preferred are polynucleotides encodingthe PDE10 polypeptide of SEQ ID NO: 2.

[0018] The disclosure of a full length polynucleotide encoding a PDE10polypeptide makes readily available to the worker of ordinary skill inthe art every possible fragment of the full length polynucleotide. Theinvention therefore provides fragments of PDE10-encoding polynucleotidesof the invention comprising at least 10 to 20, and preferably at least15, nucleotides, however, the invention comprehends fragments of variouslengths. Preferably, fragment polynucleotides of the invention comprisesequences unique to the PDE10-encoding polynucleotide sequence, andtherefore hybridize under stringent or preferably moderate conditionsonly (i.e., “specifically”) to polynucleotides encoding PDE10, or PDE10polynucleotide fragments containing the unique sequence. Polynucleotidefragments of genomic sequences of the invention comprise not onlysequences unique to the coding region, but also include fragments of thefull length sequence derived from introns, regulatory regions, and/orother non-translated sequences. Sequences unique to polynucleotides ofthe invention are recognizable through sequence comparison to otherknown polynucleotides, and can be identified through use of alignmentprograms made available in public sequence databases.

[0019] The invention also provides fragment polynucleotides that areconserved in one or more polynucleotides encoding members of the PDE10family of polypeptides. Such fragments include sequences characteristicof the family of PDE10 polynucleotides, and are also referred to as“signature sequences.” The conserved signature sequences are readilydiscernable following simple sequence comparison of polynucleotidesencoding members of the PDE10 family. Fragments of the invention can belabeled in a manner that permits their detection, and radioactive andnon-radioactive labeling are comprehended.

[0020] Fragment polynucleotides are particularly useful as probes fordetection of full length or other fragment PDE10 polynucleotides. One ormore fragment polynucleotides can be included in kits that are used todetect the presence of a polynucleotide encoding PDE 10, or used todetect variations in a polynucleotide sequence encoding PDE10.

[0021] The invention further embraces species homologs, preferablymammalian, of the human PDE10 DNA. The polynucleotide sequenceinformation provided by the invention makes possible the identificationand isolation of polynucleotides encoding related mammalian PDE10molecules by well known techniques including Southern and/or Northernhybridization, and polymerase chain reaction (PCR). Examples of relatedpolynucleotides include human and non-human genomic sequences, includingallelic variants, as well as polynucleotides encoding polypeptideshomologous to PDE10 and structurally related polypeptides sharing one ormore biological, immunological, and/or physical properties of PDE10.

[0022] The invention also embraces DNA sequences encoding PDE10 specieswhich hybridize under moderately stringent conditions to the non-codingstrands, or complements, of the polynucleotide in any one of SEQ ID NOs:1, 18, 20, and 22. DNA sequences encoding PDE10 polypeptides which wouldhybridize thereto but for the redundancy of the genetic code arecontemplated by the invention. Exemplary moderate hybridizationconditions are as follows: hybridization at 65° C. in 3× SSC, 0.1%Sarkosyl, and 20 mM sodium phosphate, pH 6.8, and washing at 65° C. in2× SSC with 0.1% SDS. Exemplary high stringency conditions would includea final wash in 0.2× SSC/0.1% SDS, at 65° C. to 75° C. It is understoodin the art that conditions of equivalent stringency can be achievedthrough variation of temperature and buffer, or salt concentration asdescribed Ausebel, et al. (Eds.), Protocols in Molecular Biology, JohnWiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridizationconditions can be empirically determined or precisely calculated basedon the length and the percentage of guanosine/cytosine (GC) base pairingof the probe. The hybridization conditions can be calculated asdescribed in Sambrook, et al., (Eds.), Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NewYork (1989), pp. 9.47 to 9.51.

[0023] Autonomously replicating recombinant expression constructionssuch as plasmid and viral DNA vectors incorporating PDE10 sequences arealso provided. Expression constructs wherein PDE10-encodingpolynucleotides are operatively-linked to an endogenous or exogenousexpression control DNA sequence and a transcription terminator are alsoprovided.

[0024] According to another aspect of the invention, host cells areprovided, including procaryotic and eucaryotic cells, either stably ortransiently transformed with DNA sequences of the invention in a mannerwhich permits expression of PDE10 polypeptides of the invention.Expression systems of the invention include bacterial, yeast, fungal,viral, invertebrate, and mammalian cells systems. Host cells of theinvention are a valuable source of immunogen for development ofantibodies specifically immunoreactive with PDE10. Host cells of theinvention are also conspicuously useful in methods for large scaleproduction of PDE10 polypeptides wherein the cells are grown in asuitable culture medium and the desired polypeptide products areisolated from the cells or from the medium in which the cells are grownby, for example, immunoaffinity purification.

[0025] Knowledge of PDE10 DNA sequences allows for modification of cellsto permit, or increase, expression of endogenous PDE10. Cells can bemodified (e.g., by homologous recombination) to provide increased PDE10expression by replacing, in whole or in part, the naturally occurringPDE10 promoter with all or part of a heterologous promoter so that thecells express PDE10 at higher levels. The heterologous promoter isinserted in such a manner that it is operatively-linked to PDE10encoding sequences. See, for example, PCT International Publication No.WO 94/12650, PCT International Publication No. WO 92/20808, and PCTInternational Publication No. WO 91/09955. The invention alsocomprehends that, in addition to heterologous promoter DNA, amplifiablemarker DNA (e.g., ada, dhfr, and the multifunctional CAD gene whichencodes carbamyl phosphate synthase, aspartate transcarbamylase, anddihydroorotase) and/or intron DNA may be inserted along with theheterologous promoter DNA. If linked to the PDE10 coding sequence,amplification of the marker DNA by standard selection methods results inco-amplification of the PDE10 coding sequences in the cells.

[0026] The DNA sequence information provided by the present inventionalso makes possible the development through, e.g. homologousrecombination or “knock-out” strategies [Capecchi, Science 244:1288-1292(1989)], of animals that fail to express functional PDE10 or thatexpress a variant of PDE10. Such animals are useful as models forstudying the in vivo activities of PDE10 and modulators of PDE10.

[0027] The invention also provides purified and isolated mammalian PDE10polypeptides as set out in SEQ ID NOs: 2, 19, 21, and 23. Presentlypreferred is a PDE10 polypeptide comprising the amino acid sequence setout in SEQ ID NO: 2. The invention embraces PDE10 polypeptides encodedby a DNA selected from the group consisting of: a) the DNA sequence setout in SEQ ID NOs:1, 18, 20, or 22; b) a DNA molecule which hybridizesunder stringent conditions to the noncoding strand of the protein codingportion of (a); and c) a DNA molecule that would hybridize to the DNA of(a) but for the degeneracy of the genetic code. The invention alsoembraces polypeptide fragments of the sequences set out in SEQ ID NOs:2, 19, 21, or 23 wherein the fragments maintain biological orimmunological properties of a PDE10 polypeptide. Preferred polypeptidefragments display antigenic properties unique to or specific for thePDE10 family of polypeptides. Fragments of the invention can be preparedby any the methods well known and routinely practiced in the art, havingthe desired biological and immunological properties.

[0028] The invention embraces polypeptides have at least 99%,at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55% and at least 50% identityand/or homology to the preferred PDE10 polypeptide on the invention.Percent amino acid sequence “identity” with respect to the preferredpolypeptide of the invention is defined herein as the percentage ofamino acid residues in the candidate sequence that are identical withthe residues in the PDE10 sequence after aligning both sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Percent sequence “homology” with respect to thepreferred polypeptide of the invention is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the residues in the PDE10 sequence after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and also considering any conservativesubstitutions as part of the sequence identity Conservativesubstitutions can be defined as set out below.

[0029] PDE10 polypeptides of the invention may be isolated from naturalcell sources or may be chemically synthesized, but are preferablyproduced by recombinant procedures involving host cells of theinvention. Use of various host cells is expected to provide for suchpost-translational modifications (e.g., glycosylation, truncation,lipidation, and phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products ofthe invention.PDE10 products ofthe invention may be full length polypeptides,biologically or immunologically active fragments, or variants thereofwhich retain specific PDE10 biological or immunological activity.Variants may comprise PDE10 polypeptide analogs wherein one or more ofthe specified (i.e., naturally encoded) amino acids is deleted orreplaced or wherein one or more non-specified amino acids are added: (1)without loss of one or more of the biological activities orimmunological characteristics specific for PDE10; or (2) with specificdisablement of a particular biological activity of PDE10.

[0030] Variant products of the invention include mature PDE10 products,i.e., PDE10 products wherein leader or signal sequences are removed, andhaving additional, non-naturally occurring, amino terminal residues.PDE10 products having an additional methionine residue at position −1(Met⁻¹-PDE10) are contemplated, as are PDE10 products having additionalmethionine and lysine residues at positions −2 and −1(Met⁻²-Lys⁻-PDE10). Variants of these types are particularly useful forrecombinant protein production in bacterial cell types.

[0031] The invention also embraces PDE10 variants having additionalamino acid residues that result from use of specific expression systems.For example, use of commercially available vectors that express adesired polypeptide such as a glutathione-S-transferase (GST) fusionproduct provide the desired polypeptide having an additional glycineresidue at position −1 as a result of cleavage of the GST component fromthe desired polypeptide. Variants which result from expression in othervector systems are also contemplated.

[0032] Variant polypeptides include those wherein conservativesubstitutions have been introduced by modification of polynucleotidesencoding polypeptides of the invention. Conservative substitutions arerecognized in the art to classify amino acids according to their relatedphysical properties and can be defined as set out in Table I (from WO97/09433, page 10, published Mar. 13, 1997 (PCT/GB96/02197, filed Sep.6. 1996). TABLE I Conservative Substitutions I SIDE CHAIN CHARACTERISTICAMINO ACID Aliphatic Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R Aromatic H F W Y Other N Q D E

[0033] Alternatively, conservative amino acids can be grouped as definedin Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc.NY:N.Y. (1975), pp.71-77] as set out in Table II. TABLE II ConservativeSubstitutions II SIDE CHAIN CHARACTERISTIC AMINO ACID Non-polar(hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T YB. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): K R H Negatively Charged (Acidic): D E

[0034] The invention further embraces PDE10 products modified to includeone or more water soluble polymer attachments. Particularly preferredare PDE 10 products covalently modified with polyethylene glycol (PEG)subunits. Water soluble polymers may be bonded at specific positions,for example at the amino terminus of the PDE10 products, or randomlyattached to one or more side chains of the polypeptide.

[0035] Also comprehended by the present invention are antibodies (e.g.,monoclonal and polyclonal antibodies, single chain antibodies, chimericantibodies, human antibodies CDR-grafted antibodies, or otherwise“humanized” antibodies, antigen binding antibody domains including Fab,Fab′, F(ab′)₂, F_(v), or single variable domains, and the like) andother binding proteins specific for PDE10 products or fragments thereofSpecific binding proteins can be developed using isolated or recombinantPDE10 products, PDE10 variants, or cells expressing such products. Theterm “specific for” indicates that the variable regions of theantibodies recognize and bind PDE10 polypeptides exclusively (i.e., ableto distinguish PDE10 polypeptides from the superfamily of PDEpolypeptides despite sequence identity, homology, or similarity found inthe family of polypeptides), but may also interact with other proteins(for example, S. aureus protein A or other antibodies in ELISAtechniques) through interactions with sequences outside the variableregion of the antibodies, and in particular, in the constant region ofthe molecule. Screening assays to determine binding specificity of anantibody of the invention are well known and routinely practiced in theart. For a comprehensive discussion of such assays, see Harlow et al.(eds), Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory;Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognizeand bind fragments of the PDE10 polypeptides of the invention are alsocontemplated, provided that the antibodies are first and foremostspecific for, as defined above, PDE10 polypeptides. As with antibodiesthat are specific for full length PDE10 polypeptides, antibodies of theinvention that recognize PDE 10 fragments are those which candistinguish PDE 10 polypeptides from the superfamily of PDE polypeptidesdespite inherent sequence identity, homology, or similarity found in thefamily of proteins.

[0036] Binding proteins are useful for purifying PDE10 products anddetection or quantification of PDE 10 products in fluid and tissuesamples using known imunological procedures. Binding proteins are alsomanifestly useful in modulating (i.e., blocking, inhibiting orstimulating) biological activities of PDE10, especially those activitiesinvolved in signal transduction. Anti-idiotypic antibodies specific foranti-PDE10 antibodies are also contemplated.

[0037] The scientific value of the information contributed through thedisclosures of DNA and amino acid sequences of the present invention ismanifest. As one series of examples, knowledge of the sequence of a cDNAfor PDE10 makes possible through use of Southern hybridization orpolymerase chain reaction (PCR) the identification of genomic DNAsequences encoding PDE10 and PDE10 expression control regulatorysequences such as promoters, operators, enhancers, repressors, and thelike. DNA/DNA hybridization procedures carried out with DNA sequences ofthe invention under moderately to highly stringent conditions arelikewise expected to allow the isolation of DNAs encoding allelicvariants of PDE10; allelic variants are known in the art to includestructurally related proteins sharing one or more of the biochemicaland/or immunological properties specific to PDE10. Similarly, non-humanspecies genes encoding proteins homologous to PDE10 can also beidentified by Southern and/or PCR analysis and useful in animal modelsfor PDE 10-related disorders. As an alternative, complementation studiescan be useful for identifying other human PDE10 products as well asnon-human proteins, and DNAs encoding the proteins, sharing one or morebiological properties of PDE10.

[0038] Polynucleotides of the invention are also useful in hybridizationassays to detect the capacity of cells to express PDE10. Polynucleotidesof the invention may also be the basis for diagnostic methods useful foridentifying a genetic alteration(s) in a PDE10 locus that underlies adisease state or states.

[0039] The DNA and amino acid sequence information provided by thepresent invention also makes possible the systematic analysis of thestructure and function of PDE10s. DNA and amino acid sequenceinformation for PDE10 also permits identification of binding partnercompounds with which a PDE10 polypeptide or polynucleotide willinteract. Binding partner compounds include proteins and non-proteincompounds such as small molecules. Agents that modulate (i.e., increase,decrease, or block) PDE10 activity or expression may be identified byincubating a putative modulator with a PDE10 polypeptide orpolynucleotide and determining the effect of the putative modulator onPDE10 phosphodiesterase activity or expression. The selectivity of acompound that modulates the activity of the PDE10 can be evaluated bycomparing its binding activity on the PDE10 to its activity on other PDEenzymes. Cell based methods, such as di-hybrid assays to identify DNAsencoding binding compounds and split hybrid assays to identifyinhibitors of PDE10 polypeptide interaction with a known bindingpolypeptide, as well as in vitro methods, including assays wherein aPDE10 polypeptide, PDE10 polynucleotide, or a binding partner areimmobilized, and solution assays are contemplated under the invention.

[0040] Selective modulators may include, for example, antibodies andother proteins or peptides which specifically bind to a PDE10polypeptide or a PDE10-encoding nucleic acid, oligonucleotides whichspecifically bind to a PDE10 polypeptide or a PDE10 gene sequence, andother non-peptide compounds (e.g., isolated or synthetic organic andinorganic molecules) which specifically react with a PDE10 polypeptideor underlying nucleic acid. Mutant PDE10 polypeptides which affect theenzymatic activity or cellular localiztion of the wild-type PDE10polypeptides are also contemplated by the invention. Presently preferredtargets for the development of selective modulators include, forexample: (1) regions of the PDE10 polypeptide which contact otherproteins and/or localize the PDE IO polypeptide within a cell, (2)regions of the PDE10 polypeptide which bind substrate, (3) cyclicnucleotide-binding site(s) of the PDE10 polypeptide, (4) phosphorylationsite(s) of the PDE10 polypeptide and (5) regions of the PDE10polypeptide which are involved in multimerization of PDE10 subunits.Still other selective modulators include those that recognize specificPDE10 encoding and regulatory polynucleotide sequences. Modulators ofPDE10 activity may be therapeutically useful in treatment of a widerange of diseases and physiological conditions in which PDE activity isknown to be involved.

[0041] PDE10 polypeptides of the invention are particularly amenable touse in high throughput screening assays to identify binding partners,and preferably modulators. Cell based assays are contemplated, includingyeast based assay systems as well as mammalian cell expression systemsas described in Jayawickreme and Kost, Curr. Opin. Biotechnol. 8:629-634(1997). Alternatively, automated and minaturized high throughputscreening (HTS)assays, such as high density free format high densityscreening, as described in Houston and Banks, Curr. Opin. Biotehcnol.8:734-740 (1997). Combinatorial libraries are particularly useful inhigh throughput screening assays.

[0042] There are a number of different libraries used for theidentification of small molecule modulators, including, (1) chemicallibraries, (2) natural product libraries, and (3) combinatoriallibraries comprised of random peptides, oligonucleotides or organicmolecules.

[0043] Chemical libraries consist of structural analogs of knowncompounds or compounds that are identified as “hits” or “leads” vianatural product screening. Natural product libraries are collections ofmicroorganisms, animals, plants, or marine organisms which are used tocreate mixtures for screening by: (1) fermentation and extraction ofbroths from soil, plant or marine microorganisms or (2) extraction ofplants or marine organisms. Natural product libraries includepolyketides, non-ribosomal peptides, and variants (non-naturallyoccurring) variants thereof For a review, see Science 282:63-68 (1998).Combinatorial libraries are composed of large numbers of peptides,oligonucleotides, or organic compounds as a mixture. They are relativelyeasy to prepare by traditional automated synthesis methods, PCR, cloningor proprietary synthetic methods. Of particular interest are peptide andoligonucleotide combinatorial libraries. Still other libraries ofinterest include protein, peptidomimetic, multiparallel syntheticcollection, recombinatorial, and polypeptide libraries. For a review ofcombinatorial chemistry and libraries created therefrom, see Myers,Curr. Opion. Biotechnol. 8:701-707 (1997). Identification of modulatorsthrough use of the various libraries described herein permitsmodification of the candidate “hit” (or “lead”) to optimize the capacityof the “hit” to modulate activity.

[0044] Also made available by the invention are anti-sensepolynucleotides which recognize and hybridize to polynucleotidesencoding PDE10. Full length and fragment anti-sense polynucleotides areprovided. The worker of ordinary skill will appreciate that fragmentanti-sense molecules of the invention include (i) those whichspecifically recognize and hybridize to PDEIO RNA (as determined bysequence comparison of DNA encoding PDE10 to DNA encoding other knownmolecules) as well as (ii) those which recognize and hybridize to RNAencoding variants in the PDE10 family of proteins. Antisensepolynucleotides that hybridize to RNA encoding other members of thePDE10 family of proteins are also identifiable through sequencecomparison to identify characteristic, or signature, sequences for thefamily of molecules. Anti-sense polynucleotides are particularlyrelevant to regulating expression of PDE10 by those cells expressingPDE10 mRNA.

[0045] Antisense nucleic acids (preferably 10 to 20 base pairoligonucleotides) capable of specifically binding to PDE 10 expressioncontrol sequences or PDE10 RNA are introduced into cells (e.g., by aviral vector or colloidal dispersion system such as a liposome). Theantisense nucleic acid binds to the PDE10 target nucleotide sequence inthe cell and prevents transcription or translation of the targetsequence. Phosphorothioate and methylphosphonate antisenseoligonucleotides are specifically contemplated for therapeutic useaccording to the invention. The antisense oligonucleotides may befurther modified by poly-L-lysine, transferrin polylysine, orcholesterol moieties at the 5′ end.

[0046] The invention further comprehends methods to modulate PDE10expression through use of ribozymes. For a review, see Gibson andShillitoe, Mol. Biotech. 7:125-137 (1997). Ribozyme technology can beutilized to inhibit translation of PDE10 mRNA in a sequence specificmanner through (i) the hybridization of a complementary RNA to a targetmRNA and (ii) cleavage of the hybridized mRNA through nuclease activityinherent to the complementary strand. Ribozymes can identified byempirical methods but more preferably are specifically designed based onaccessible sites on the target mRNA [Bramlage, et al., Trends in Biotech16:434-438 (1998).] Delivery of ribozymes to target cells can beaccomplished using either exogenous or endogenous delivery techniqueswell known and routinely practiced in the art. Exogenous deliverymethods can include use of targeting liposomes or direct localinjection. Endogenous methods include use of viral vectors and non-viralplasmids.

[0047] Ribozymes can specifically modulate expression of PDE10 whendesigned to be complementary to regions unique to a polynucleotideencoding PDE10. “Specifically modulate” therefore is intended to meanthat ribozymes of the invention recognizes only a polynucleotideencoding PDE10. Similarly, ribozymes can be designed to modulateexpression of all or some ofthe PDE10 family of proteins. Ribozymes ofthis type are designed to recognize polynucleotide sequences conservedin all or some of the polynucleotides which encode the family ofproteins.

[0048] The invention further embraces methods to modulate transcriptionof PDE10 through use of oligonucleotide-directed triplet helixformation. For a review, see Lavrovsky, et al., Biochem. Mol. Med.62:11-22 (1997). Triplet helix formation is accomplished using sequencespecific oligonucleotides which hybridize to double stranded DNA in themajor groove as defined in the Watson-Crick model. Hybridization of asequence specific oligonucleotide can thereafter modulate activity ofDNA-binding proteins, including, for example, transcription factors andpolymerases. Preferred target sequences for hybridization includepromoter and enhancer regions to permit transcriptional regulation ofPDE10 expression. Oligonucleotides which are capable of triplet helixformation are also useful for site-specific covalent modification oftarget DNA sequences. Oligonucleotides useful for covalent modificationare coupled to various DNA damaging agents as described in Lavrovsky, etal. [supra].

[0049] The invention comprehends mutations in the PDE10 gene that resultin loss of normal function of the PDE10 gene product and underlie humandisease states in which failure ofthe PDE10 is involved. Gene therapy torestore PDE10 activity would thus be indicated in treating those diseasestates. Delivery of a functional PDE10 gene to appropriate cells iseffected ex vivo, in situ, or in vivo by use of vectors, and moreparticularly viral vectors (e.g., adenovirus, adeno-associated virus, ora retrovirus), or ex vivo by use of physical DNA transfer methods (e.g.,liposomes or chemical treatments). See, for example, Anderson, Nature,supplement to vol. 392, no. 6679, pp.25-20 (1998). For additionalreviews of gene therapy technology see Friedmann, Science, 244:1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller,Nature, 357: 455-460 (1992). Alternatively, it is contemplated that inother human disease states, preventing the expression of or inhibitingthe activity of PDE10 will be useful in treating the disease states. Itis contemplated that antisense therapy or gene therapy could be appliedto negatively regulate the expression of PDE10.

[0050] Identification of modulators of PDE10 expression and/orbiological activity provides methods to treat disease states that arisefrom aberrant PDE10 activity. Modulators may be prepared in compositionsfor administration, and preferably include one or more pharmaceuticallyacceptable carriers, such as pharmaceutically acceptable (i.e., sterileand non-toxic) liquid, semisolid, or solid diluents that serve aspharmaceutical vehicles, excipients, or media. Any diluent known in theart may be used. Exemplary diluents include, but are not limited to,polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- andpropylhydroxybenzoate, talc, alginates, starches, lactose, sucrose,dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineraloil, cocoa butter, and oil of theobroma. The modulator compositions canbe packaged in forms convenient for delivery. The compositions can beenclosed within a capsule, sachet, cachet, gelatin, paper, or othercontainer. These delivery forms are preferred when compatible with entryof the composition into the recipient organism and, particularly, whenthe composition is being delivered in unit dose form. The dosage unitscan be packaged, e.g., in tablets, capsules, suppositories or cachets.The compositions may be introduced into the subject by any conventionalmethod including, e.g., by intravenous, intradermal, intramuscular,intramammary, intraperitoneal, or subcutaneous injection; by oral,sublingual, nasal, anal, vaginal, or transdermal delivery; or bysurgical implantation, e.g., embedded under the splenic capsule or inthe cornea. The treatment may consist of a single dose or a plurality ofdoses over a period of time.

[0051] The invention also embraces use of a PDE10 polypeptide, a PDE10polynucleotide, or a binding partner thereof in production of amedicament for treatment of a PDE10-related biological disorder.

[0052] The present invention is illustrated by the following examplesrelating to the isolation of a polynucleotide encoding a PDE10polypeptide and expression thereof Example 1 describes identification ofan EST encoding a partial PDE10 polypeptide and isolation of a fulllength PDE10-encoding clone. Example 2 relates to Northern blot analysisof PDE10 expression. Example 3 addresses chromosome mapping of PDE10.Example 4 describes expression and characterization of a recombinantPDE10 polypeptide. Example 5 describes production of anti-PDE10antibodies. Example 6 provides an analysis of PDE 10 expression using insitu hybridization. Example 7 relates to high throughput screening toidentify inhibitors of PDE10.

EXAMPLE 1 Identification of an EST Related to a Human PDE and Isolationof a Full Length PDE10-Encoding Polynucleotide

[0053] Using the sequences of known human, 3′, 5′ cyclic nucleotidephosphodiesterases, a search of the National Center for BiotechnologyInformation (NCBI) Expressed Sequence Tags (EST) database was undertakenin order to identify cDNA fragments that could potentially be useful forthe identification of novel phosphodiesterase (PDE) genes. This databasecontains DNA sequences representing one or both ends of cDNAs collectedfrom a variety of tissue sources. A single sequencing run is performedon one or both ends of the cDNA and the quality of the DNA sequencevaries tremendously. At the time the PDE searches were performed, theEST sequence database contained more than 600,000 cDNA sequences from avariety of organisms.

[0054] The search for novel PDE sequences included three steps. First,the BLASTN program available through NCBI was used to identify DNAsequences in the EST sequence database with homology to cDNA sequencesencoding known human PDEs. The program compares a nucleotide querysequence against a nucleotide sequence database. The cDNA sequences ofthe fifteen known human PDEs were submitted and fifteen BLASTN searcheswere performed; the query PDE sequences included PDE1A3 [Loughney, etal., J. Biol. Chem. 271:796-806 (1996)], PDE1B1 [Yu, et al., CellSignaling, 9:519-529 (1997)], PDE1C2 [Loughney, et al., J. Biol. Chem.271:796-806 (1996)], PDE2A3 [Rosman, et al., Gene 191:89-95 (1997)],PDE3A [Meacci, et al., Proc. Natl. Acad. Sci. (USA) 89:3721-3725(1992)], PDE3B [Miki et al, Genomics 36:476-485 (1996)], PDE4A5[Bolger,et al., Mol. Cell. Biol. 13:6558-6571 (1993)], PDE4B2 [Bolger,et al., Mol. Cell. Biol. 13:6558-6571 (1993)], PDE4C [Bolger, et al,Mol. Cell Biol. 13:6558-6571 (1993)], PDE4D1 [Bolger, et al., Biochem.J. 328:539-548 (1997)] and PDE4D3 [Bolger, et al., Mol. Cell. Biol.13:6558-6571 (1993)], PDE5A, PDE6A [Pittler, et al., Genomics 6:272-283(1990)], PDE6B [Collins, et al., Genomics 13:698-704 (1992)], PDE6C[Piriev, et al., Genomics 28:429-435 (1995), and PDE7A1 [Michaeli, etal., J. Biol. Chem. 17:12925-12932 (1993)]. The BLASTN results wereexamined and EST sequences that were judged as corresponding to each ofthe fifteen known PDE cDNAs were identified and collected into a table.The PDE6A and PDE6B sequences used as queries were truncated at 3′ end(removing a portion of the 3′ untranslated region) due to the presenceof repetitive elements in the 3′ untranslated region of the cDNAs.

[0055] Secondly, the NCBI TBLASTN program was used to examine thehomology between the protein sequence of the fifteen known human PDEs(as above) and the six different possible proteins encoded by each ofthe EST DNA sequences. In this search, the EST sequences are translatedin the six possible reading frames and the amino acid sequencesgenerated are compared to the query PDE amino acid sequences. Sequencesidentified as homologous at the amino acid level were examined and anyEST sequences positively identified as corresponding to a known PDEduring the BLASTN search described above were discarded.

[0056] The third step of the search involved analyzing the sequencesthat were not known PDEs. These amino acid sequences were homologous toa known PDE but were not identified as one of the 15 known PDE genesduring the BLASTN searches.

[0057] The initial BLAST searches identified three EST sequences,designated X88347 (SEQ ID NO: 3), X88467 (SEQ ID NO: 4), and X88465 (SEQID NO: 5), that were obtained from an exon trapping experiment usingchromosome 21 genomic DNA and found to encode an amino acid sequencehaving homology to the catalytic region of one or more of the PDE querysequences. X88347 showed homology with the amino acid sequences ofPDE1A, 1B, 1C, 3A, 3B, 4A, 4B and 4D; X88467 showed homology to PDE1A,1B, 1C, 4A 4B, 4C, and D4; and X88465 was homologous to PDE1A and 1Bamino acid sequences. At the 5′ terminus, EST X88465 was 58 nucleotidesshorter than was X88467 and was not considered further.

[0058] When X88347 was translated from nucleotides 1-222 and theresultant protein was compared to PDE1A, the two proteins were the sameat 23 of 51 amino acid positions (45% identity). When X88467 wastranslated from nucleotide 3 to 155 and the resultant protein comparedto PDE1A, 15 of 36 amino acids were the same (42% identity). BecauseESTs X88347 and X88467 showed homology to two different regions of thecatalytic region of PDE1A, it seemed possible that they represented twodifferent exons from a novel PDE gene.

[0059] X88347 was used as a query in a BLASTN search of the NCBI ESTdatabase. In addition to itself, X88347 identified three other human ESTsequences with high enough homology to suggest the sequences werederived from the same gene. EST R00718 (SEQ ID NO: 6) showed 91%identity to X88347. R00719 (SEQ ID NO: 7) represented the 3′-end of thesame cDNA as R00718. R45187 (SEQ ID NO: 8) showed 88% identity toX88347. Two mouse cDNAs were also identified; W82786 (SEQ ID NO: 9)(91%identity) and W10517 (SEQ ID NO: 10) appeared to represent the mousehomolog of X88347. A BLASTN search using W10517 as probe identifiedanother sequence H90802 (SEQ ID NO: 11), which appeared to representanother human EST that may be part of the human PDE gene. The severalhuman cDNAs were not identical to each other, and the quality of thesequencing was poor. The cDNA represented by the R00719 and R00718 ESTsequences was obtained from the American Type Culture Collection(Rockville, Md.) which maintains and makes publicly available depositsof ESTs identified and sequenced by I.M.A.G.E., Lawrence LivermoreNational Laboratory, (Livermore, Calif.). The cDNA had been isolatedfrom a fetal liver and spleen library and mapped to chromosome 21.

[0060] R00718/9 was sequenced upon receipt and found to be consistentwith the EST database sequence. The polynucleotide and amino acidssequences for R00718/9 are set out in SEQ ID Nos: 12 and 13,respectively. The R00718/9 clone contained a 0.6 kb insert with a poly Atail at the 3′-end. The open reading frame encoded a protein withhomology to other PDEs but did not extend to the 5′end of the cDNA.Beginning at amino acid position 9, a QSDRE sequence was found.Corresponding D and E residues were found within all of the querysequences. Query sequences also included a conserved E(F/Y) sequencelocated amino terminal to the conserved D and E residues, but thissequence was not found in EST R00718/9. Instead, the EST contained eightamino acids followed by a stop codon. The R00718/9 cDNA appeared todiverge from the PDE query sequences in the catalytic region and theopen reading frame was not maintained. The disrupted open reading framemay suggest the presence of an intron that had not been removed or thatthe R00718/9 sequence was joined to some unidentified extraneouspolynucleotide sequence. The gene represented by R00718/9 was designatedPDE10.

[0061] In order to identify additional PDE10 sequences, a probe wasgenerated based on the PDE10 sequence and used to screen cDNA libraries.First, two primers, R71S100R (SEQ ID NO: 14) and R71A521H (SEQ ID NO:15) were synthesized for use in PCR to amplify a 420 nucleotide portionof the R00718/9 DNA fragment (nucleotides 130 to 550). Primer R71S100Rgenerated anEcoRI restriction site in the amplification product(underlined below) and primer R71A521H generated a HindIII site (alsounderlined below). The PCR fragment was designed to include the regionof R00718/9 homologous to other PDEs, but not the poly A tail. R71S100R(SEQ ID NO:14) AGTCGAATTCACCGTGAGAAGTCAGAAG R71A521H (SEQ ID NO:15)GTCAAAGCTTACATGGTCTTGTGGTGCC

[0062] The PCR reaction contained 50 pg R00719 cDNA, 10 ng/μl eachprimer, 0.2 mM dNTP, 1× PCR buffer (Perkin-Elmer), 2 mM MgCl₂, and 1.25U Taq polymerase (Perkin-Elmer). The reaction was first maintained at94° C. for four minutes, after which thirty cycles of one minute 94° C.,two minutes 50° C., and four minutes at 72° C. were performed. The PCRfragment was purified using low melting point agarose gelelectrophoresis.

[0063] For library screening, the PCR fragment was labeled with ³²P witha random priming kit (Boehringer Mannheim) according to manufacturer'sinstructions and used to screen 10⁶ cDNAs from a human heart cDNAlibrary (Stratagene, La Jolla, Calif.), 5×10⁵ cDNAs from a humanhippocampal cDNA library (Clontech, Palo Alto, Calif.), and 7.5×10⁵cDNAs from a human fetal brain cDNA library (Stratagene). Hybridizationwas carried out overnight in buffer containing 3× SSC, 0.1% Sarkosyl, 20mM sodium phosphate, pH 6.8, 10× Denhardt's solution, and 50 μg/mlsalmon sperm DNA at 65° C. Eleven positives were obtained from the fetalbrain library and three from the hippocampal library. Partial sequencingled to the selection of one, FB79c, for further characterization. Thepolynucleotide and deduced amino acid sequences for FB79c are set out inSEQ ID NOs: 16 and 17, respectively.

[0064] FB79c contained a 1.3 kb insert; the 3′end of FB79c extendedfurther than that of R00718/9 and contained 12 adenosine residues of thepoly A tail of R00718/9, an EcoRI site (GGAATTC), an additionalfifty-nine nucleotides and a poly A sequence. At the 5′end, the sequencefor FB79c differed from that of R00718/9 beginning at, and continuing 5′of, nucleotide 121 of R00718/9 (corresponding to nucleotide 744 ofFB79c). The open reading frame in FB79c (encoding a protein withhomology to the query PDEs) did not extend to the 5′end of the cDNA butended in a stop codon at nucleotide 104.

[0065] A sequence within the FB79c DNA located upstream of the point ofdivergence from R00718/9 (but within the portion of the open readingframe with homology to the other PDEs) was the region chosen for a probein subsequent library screening. The isolated sequence selected was a0.36 kb EcoRV fragment extending from nucleotide 308 to nucleotide 671of FB79c and was used to screen 1.75×10⁶ cDNAs from the fetal brain cDNAlibrary (Stratagene). More than twenty cDNAs were identified and twelvewere subjected to partial restriction mapping and DNA sequencing. Moreextensive sequencing on six of them led to the selection of clonesFB76.2 and FB68.2 for complete sequencing. The polynucleotide and aminoacid sequences for clone FB76.2 are set out in SEQ ID NOs: 18 and 19,respectively, and the polynucleotide and amino acid sequences for cloneFB68.2 are set out in SEQ ID NOs. 20 and 21, respectively.

[0066] FB76.2 contained a 1.9 kb cDNA insert; the 3′end of the cDNAstopped one nucleotide short of the poly A tail found in clone FB79c andthe sequence diverged from FB79c 5′ of nucleotide 109 in clone FB79c(corresponding to nucleotide 715 in FB76.2). The open reading frame inthe FB76.2 sequence that encoded a protein with homology to the PDEquery sequences extended to the 5′end of the cDNA and the firstmethionine was encoded beginning at nucleotide 74. Assuming this residueto be the initiating methionine, the open reading frame of FB76.2encoded a 533 amino acid protein with a predicted molecular weight of61,708 Da.

[0067] Clone FB68.2 contained a 2 kb CDNA insert. At the 3′end, itextended to the poly A tail found in the FB79c sequence and the openreading frame extended to the 5′end of the CDNA. FB68.2 differed fromFB76.2 by the presence of an additional internal 180 nucleotides(nucleotides 225 to 404 of FB68.2) following corresponding nucleotide335 of FB76.2. Since the number of additional nucleotides in the FB68.2insertion was divisible by three, it did not alter the reading frame ascompared to FB76.2. The position of the insert with respect tomaintaining the same reading frame suggested that the sequence mightrepresent an exon found in some, but not all, PDE10 cDNAs.Alternatively, the additional sequence could be an intron that had notbeen removed from the FB68.2 cDNA.

[0068] Because the FB76.2 and FB68.2 differed from each other,additional PDE10 DNAs were obtained and analyzed to more accuratelydefine the PDE10 nucleotide sequence. A 5′ 0.3 kb EcoRI fragment ofFB76.2 (corresponding to nucleotides 1 to 285) was isolated and used asa probe to screen 7.5×10⁵ cDNAs from the fetal brain cDNA library.Thirty seven positives were obtained, of which nineteen were firstcharacterized with respect to fragment size (insert) that hybridized tothe 0.3 kb EcoRi probe. Eight of the nineteen clones were subsequentlycharacterized by partial sequencing. Two clones, FB93a and FB94a,contained 0.5 kb and 1.6 kb EcoRI fragments, respectively, thathybridized and were chosen for complete sequencing. The polynucleotideand amino acid sequences for clone FB93a are set out in SEQ ID NOs: 22and 23, respectively, and the polynucleotide and amino acid sequencesfor clone FB94a are set out in SEQ ID NOs: 1 and 2, respectively.

[0069] FB93a contained a 1.5 kb insert which did not extend to the 3′endof FB76.2 but was ninety nucleotides longer than FB76.2 at the 5′end.The additional nucleotides encoded a stop codon beginning at position 47which was in reading frame with the first methionine in FB76.2 describedabove (nucleotide 164 in FB93a). The position of the stop codonindicated the presence of a complete open reading frame and that FB76.2probably represented a full length cDNA. Like FB76.2, FB93a did notcontain the 180 nucleotide insert that was present in FB68.2.

[0070] FB94a contained a 1.5 kb cDNA insert and the 3 ′end extendedalmost 0.1 kb beyond the stop codon. The first methionine was encodedbeginning at nucleotide 26, and assuming this residue to be theinitiating methionine, FB94a encoded a 466 amino acid protein with apredicted molecular weight of 54,367 Da. FB94a differed from FB76.2 andFB93a by absence of a 149 nucleotide region which, if consistent withthe sequences for FB76.2 and FB93a, would have been located afternucleotide 42. The absence of the 149 nucleotide sequence produced aputative initiator methionine that is in a different reading frame thanthat found in FB76.2 and FB93a. Like FB76.2 and FB93a, FB94a did notcontain the 180 nucleotide region found in FB68.2.

[0071] A search of the EST data base with the FB94a and FB93a sequencesidentified yet another possible sequence for a PDEIO cDNA. The sequenceof EST A158300 lacked both the 149 nucleotide and the 180 nucleotidesequences discussed above. In addition, A158300 also lacked 55nucleotides immediately 3′ to the 180 nucleotide region as found in theFB68.2 sequence. The open reading frame in A158300 extended to the 5′endand the first methionine corresponded to the same one used by FB76.2 andFB93a. The presence of the additional 55 nucleotide deletion fromA158300 resulted in a different reading frame for the sequence betweenthe site where the 149 nucleotides were deleted and the site where the180 nucleotides were deleted.

[0072] The sequence information for PDE10 derived from these cDNAsequences can be summarized as follows. There is a 149 nucleotidesequence found in some clones (sequences FB76.2, FB93a, FB68.2) but notin all (sequences FB94a, A158300). The 149 nucleotide sequence isfollowed by a 44 nucleotide region that is present in all the PDE10cDNAs analyzed to date. Following the 44 nucleotide region is a sequenceof 235 nucleotides in length. The region can be present in its entirety(as found in the sequence for FB68.2) or without the first 180nucleotides (as observed in sequences FB76.2 and FB93a). As stillanother alternative, the whole region can be removed (as found in thesequence for A158300). These possibilities predict six different mRNAstructures, four of which have been isolated.

[0073] The presence or absence of the 149 nucleotide region may reflectthe presence or absence of an exon, and the presence of all or some ofthe 235 nucleotide region may reflect alternative 3′splice acceptor siteusage. As an alternative, it is also possible that the 235 nucleotideregion represents two separate exons of 180 and 55 nucleotides inlength. The presence or absence of the 149 nucleotide sequence altersthe reading frame of the encoded protein as does the presence or absenceof the 55 nucleotide sequence.

[0074] A number of single nucleotide differences have been observed incomparison of the various PDE10 cDNAs. R00718/9 has a cytosine atnucleotide position 155 whereas the other cDNAs have a thymidine at thisposition; this difference represented a silent change as proline isencoded by both sequences. R00718/9 also has a cytosine at position 161whereas the other cDNAs have an adenosine at the same position; thisdifference also represented a silent change as both sequences encodealanine. FB94a has a guanosine at position 1383 whereas the other cDNAshave an adenosine at this position; as a consequence of the difference,FB94a encodes a glycine rather than a glutamic acid at that position.FB76.2 has an adenosine rather than a cytosine at position 1809; thedifference does not effect an amino acid difference since the nucleotideposition is located in the 3′ untranslated region. FB79c also has oneless adenosine in the string of nucleotides between 1204 and 1215 thando the other cDNAs; this difference is also within the 3′ untranslatedregion.

[0075] In comparison of a predicted PDE10 amino acid sequence with otherknown PDEs indicated that most, but not all, of the amino acids that areconserved among the query sequences were also found in PDE10. Comparisonof the PDE10 catalytic region to PDE4A, PDE5A, and PDE7A revealed 32%,30% and 34% identity, respectively.

EXAMPLE 2 Northern Blot

[0076] In order to determine which cell and tissue types express PDE10,Northern blot analysis was carried out using a commercially preparedmulti-tissue Northern blot (Clontech, Palo Alto, Calif.). The probe wasa EcoRI/BclI fragment of the FB76.2 corresponding to nucleotides 1 to883. Hybridization conditions were as previously described [Loughney etal., supra, (1996)].

[0077] Results indicated a 2.2 to 2.4 kb band which was strongest inkidney, present in heart, pancreas, and placenta, and weakest in brain,lung, skeletal muscle and liver. The band was fairly wide in placentasuggesting that it might contain a number of mRNAs of slightly differentsizes

EXAMPLE 3 Chromosome Mapping

[0078] As mentioned above, the X88347, X88467, and X88465 ESTs wereidentified with an exon trapping procedure using DNA from chromosome 21[Chen et al. 1996]. X88467 was identified as a new sequence withhomology to a mouse calcium-, calmodulin-dependent phosphodiesteraseQ01065 aa 52-103. XD88347 was identified to be the same as EST R00718and similar to Drosophila cAMP dependent phosphodiesterase P12252. Bothof these sequences were placed in a category described as having stronghomology to known protein sequences.

[0079] A search of the Sequence Tagged Sites (STS) database at NCBIrevealed homology of the 3′-end of PDEIO to STS WI-13322 which has beenmapped to region 220.72 cr. from the top of chromosome 21. The cDNA thatthis STS was derived from begins at nucleotide 1899 of FB68.2, does nothave the poly A tail and extends further 3′ than FB68.2. It seems likelythat this STS sequence represents a PDE10 A transcript to which no poly(A+) tail has been added or a PDE10A transcript that uses an alternativesite for poly (A+) addition. STS WI-13322 was placed on a Whitehead mapof chromosome 21 near SGC35805, which is derived from the gene for thecystathionine beta-synthase (CBS). CBS has been mapped to chromosome 21at 21q22.3 [Avramopoulos, et al, Hum. Genet. 90:566-568 (1993); Munke etal., Hum. Genet. 42:550-559 (1988)].

[0080] A number of different genetic diseases map to this region ofchromosome 21, for example, Down syndrome [Delabar, et al., Eur. J. Hum.Genet. 1:114-124(1993)]. It is not clear that PDE10A falls within theDown syndrome critical region (DSCR) but it is possible that geneselsewhere on chromosome 21 also contribute to Down syndrome [Korenberg,et al., Proc. Natl. Acad. Sci. (USA) 91:4997-5001 (1994)]. As anotherexample, a locus involved in bipolar affective disorder in some familieshas been mapped to 21 q22.3 [Vallada, et al., J. Affect. Disord.41:217-221 (1996)]. Other examples include Knobloch syndrome,characterized by myopia and retinal degeneration and detachment [Sertie,et al., Hum. Mol. Genet. 5:843-847 (1996)], and one or more genesresponsible for congenital recessive deafness (DFNB8, DFNB10) [Veske, etal., Hum. Mol. Genet. 5:165168 (1996); Bonne-Tamir, et al., Am. J. Hum.Genet. 58:1254-1259 (1996)]. PDE10A may play a role in any or all ofthese disease states.

EXAMPLE 4 Expression and Characterization of PDE10

[0081] The entire open reading frame of the PDE10 cDNA (clone FB94a) wasplaced into a yeast ADH vector including the alcohol dehydrogenasepromoter. The construct was built in two steps.

[0082] The 5′end was generated using PCR and FB94a DNA as template. PCRwas carried out using the 5′ primer below (SEQ ID NO: 25) in combinationwith 3′ primer R71A3 (SEQ ID NO: 26). The 5′ primer includes an NcoIsite (underlined in SEQ ID NO: 25 below) and the initiating methioninecodon of FB94a is in bold. The 5′ primer also adds a FLAG® epitope tag(Eastman Kodak, Rochester, N.Y.) to the amino terminus of the encodedprotein; the FLAG® tag is an epitope (SEQ ID NO: 24) recognized by themonoclonal antibody M2 (Eastman Kodak). FLAG®TAG (SEQ ID NO:24)Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys 5′ Primer (SEQ ID NO:25)TAGACCATGGACTACAAGGACGACGA- TGACAAGATGGACGCATTCAGAAGCACT R71A3 (SEQ IDNO:26) CGAGGAGTCAACTTCTTG

[0083] PCR was carried out using 5 μl each primer (100 μg/ml stock), 5μl 10× buffer (Perkin Elmer), 5 μl 10× nucleotides (2 mM stock), 3 μlMgCl₂ (25 mM stock), FB94a DNA, and 0.3 μl Taq polyrnerase (PerkinElmer) in a reaction volume of 50 μl. After incubating the reactionmixture at 94° C. for four minutes, 30 cycles of one minute at 94° C.,two minutes at 50° C., and four minutes at 72° C. were carried out. ThePCR product was cleaved with NcoI and HincII and purified using agarosegel electrophoresis. The 3′sequence of PDE10 was isolated as aHincII/EcoRI fragment cleaved from FB94a and purified by agarose gelelectrophoresis. The two fragments were combined and ligated into aNcoI/EcoRI-digested Bluescript® vector (Stratagene, La Jolla, Calif.),previously modified by the insertion of the ADH promoter previouslyremoved from a YEpC-PADH2d vector [Price et al. Meth. Enzymol.185:308-315 (1990)] as a SacI/NcoI fragment, to generate plasmidPDE10-1. New junctions and sequence generated by PCR were verified bysequencing.

[0084] In the second step of plasmid construction, the SacI/SalIfragment from PDE10-1 containing the ADH promoter and PDE10 open readingframe was purified by two rounds of agarose gel electrophoresis andligated into SacI/SalI cut YEpC-PADH2 vector.

[0085] Following transformation into BJ2-54, a yeast strain lackingendogenous PDE activity, a colony was selected, streaked out on SC-leuplates and a single colony carrying the PDE10 construct was chosen forfurther characterization. Following overnight growth in SC-leu media theculture was diluted 1:250 in fresh SC-leu and grown overnight at 30° C.until it reached a density of 10⁷ cells/ml. The cells were collected bycentrifugation, washed once with YEP 3% glycerol media, resuspended inYEP containing 3% glycerol, and grown at 30° C. for another 24 hours.The cells were harvested by centrifugation, washed with water, andfrozen at −70° C. until use. Prior to use, an aliquot of the yeastextract was analyzed by SDS PAGE. A protein specific to yeast carryingthe PDE10 expression construct that migrated on the SDS PAGE gels withthe expected mobility (55.5 kDa) was observed by Coomassie bluestaining.

[0086] Yeast cells ( 1×10¹⁰) were thawed with 200 μg/ml each ofpepstatin A, leupeptin, and aprotinin 1 mM DTT, and 20 μg/ml calpaininhibitors (I and II). Two hundred μl of glass beads (0.5 mm, acidwashed) were added, and the mixture was vortexed for eight cycles of 30seconds each. Samples were cooled for 4.5 minutes at 4° C. betweencycles. After lysis, 0.8 ml lysis buffer was added, the lysate separatedfrom the beads, and the lysate centrifuged for 30 minutes at 100,000 ×gin a Beckman TL-100 tabletop centrifuge. The supernatant was aliquoted,frozen in dry ice/ethanol, and stored at −70° C.

[0087] Kinetic assays were performed on a BIOMEK® 1000 programmablerobotic station (Beckman Instruments). The range of final substrateconcentration was 0.2 to 1000 μM for cAMP and 0.6-2000 nM for cGMP. Thehighest nucleotide concentration contained 1 to 1.5 million Cerenkovcounts of ³²P-labeled substrate per assay. The enzyme preparation wasinitially diluted 1:500 (cAMP as substrate) or 1:50,000 (cGMP assubstrate). The enzyme dilution buffer consisted of 25 mM Tris-HCl pH8.9, 5 μM ZnSO₄ 5 mM MgCl₂, 0.1 mM DTT, 100 mM NaCl and 0.1 mg/ml BSA(Calbiochem; fatty acid free). Activity at each substrate concentrationwas derived from a linear fit of successive four-fold enzyme dilutionsacross the plate.

[0088] Assays were performed at 30° C. for 15 minutes. After 12 minutes,5 μl snake venom from Crotalus atrox (15 mg/ml protein) was added toeach reaction. Assays were stopped by addition 200 μl of charcoalsuspension (25 mg/ml activated charcoal in 0.1 M monobasic potassiumphosphate). The plate was centrifuged at 2600 rpm, and 200 μl of eachsupernatant was transferred into Microbeta® counting plates and countedon a WALLAC Microbeta® by Cerenkov counting. Data were evaluated with apredesigned Microsoft Excel® Spreadsheet, and the kinetic parameterswere fitted to a Michaelis-Menton model using the program Table Curve®from Jandel Scientific.

[0089] Results indicated that the K_(m) for cGMP hydrolysis was 5 (±1)nM and the K_(m) for cAMP hydrolysis was 160 (±30) μM. In the extract,cGMP hydrolytic activity was determined to be 0.035 (±0.01) μmol/min/mg,while cAMP hydrolysis was measured to be 0.52 (±0.06) μmol/min/mg. Thus,although PDE10 had much greater affinity for cGMP, the V_(max) for cAMPwas 15-fold greater.

[0090] In order to distinguish PDE10 from other PDE families, a panel ofPDE inhibitors with activities against defined PDE families was testedfor PDE10 inhibition using cAMP as a substrate. The results of the assayare set out in Table 1 below. TABLE 1 PDE10 Inhibition withIsozyme-specific PDE Inhibitors Target PDE10 Target Family InhibitorFamily IC₅₀ (μM) IC₅₀ (μM) SCH46642 PDE1 14 0.2⁵ EHNA PDE2 477 0.8²Cilostamide PDE3 100 0.04-0.9³ Rolipram PDE4 529 0.18-0.5⁴ DMPPO PDE5 90.003¹ IBMX non-specific 59  2-20¹

[0091] The results further distinguish PDE10 from PDEs in families 1through 5 in that specific inhibitors for enzymes in those families aresignificantly less effective in inhibiting PDE10.

EXAMPLE 5 Production of Anti-PDE10 Antibodies

[0092] A GST fusion protein was produced in E. coli to provide anantigen for generation of monoclonal antibodies to PDE10. An EcoRIfragment from FB76.2 (nucleotides 280 through 1829 in SEQ ID NO: 18) wasinserted into the EcoRI site of pGEX3X (Pharmacia) and the resultantconstruct was transformed in the E. coil strain XLI Blue. A GST-PDE10fusion protein including 464 amino acids from PDE10 was expressed fromthis construct following induction with IPTG. The fusion protein wasisolated using SDS-PAGE, the band of appropriate size excised from thegel following staining with cold 0.4 M KCl, and the protein obtainedfrom the acrylamide by electroelution. The elution product was dialyzedagainst PBS and concentrated using Centriprep 10 and Centricon columns(Arnicon, Beverly Mass.) prior to being injected into mice.

[0093] On day 0, four Balb/c mice were pre-bled and immunized bysubcutaneous injection with a panel of antigens including 30 μg/mouseGST-PDE10 fusion protein in complete Freund's adjuvant in 200 μl totalvolume. The same injections were repeated at weeks three and nine inincomplete Freund's adjuvant. Ten days after the last immunization, testbleeds were obtained and screened by antigen capture ELISA and Westernanalysis.

[0094] In the ELISA, Immulon® 4 plates (Dynex, Cambridge, Mass.) werecoated at 4° C. with 50 μl/well of a solution containing 2 μg/mlGST-PDE10 in 50 mM carbonate buffer, pH 9.6. Plates were blocked with0.5% fish skin gelatin (Sigma) for 30 minutes and 50 μl serum diluted inPBS with 0.5% Tween® 20 (PBST) was added. Serum dilutions ranged from1:100 to 1:102,400 and were obtained by a series of doubling dilutions.After incubation at 37° C. for 30 minutes and washing three times withPBST, 50 μl of horseradish peroxidase-conjugated goat anti-mouse IgG(fc)antibody (Jackson) (diluted 1:10000 in PB ST) was added. Plates wereincubated as above and washed four times with PBST. Antibody wasdetected with addition of tetramethyl benzidine (Sigma Chemical, St.Louis, Mo.) and the color reaction was stopped after five minutes withthe addition of 50 μl of 15% H₂SO₄. Absorbance at 450 nM was measured ona plate reader.

[0095] For Western analysis, SDS-PAGE gels were run with approximately10 μg yeast PDE10 extract and approximately 200 ng of gel-purifiedGST-PDE10 and the proteins were transferred to Immobilon-PVDF. Astandard enhanced chemiluminescence (ECL) Western blot protocol wasperformed using BioRad goat anti-mouse IgG horseradish peroxidase as thesecondary antibody.

[0096] In preparation of hybridomas, splenocytes from mice giving apositive result from the ELISA and/or Western blotting protocols above,were fused to NS-1 cells in a ratio of 5:1 by standard methods usingpolyethylene glycol 1500 (Boehringer Mannheim) [Harlow and Lane,Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory, p.211(1988)]. The fused cells were resuspended in 200 ml RPMI containing 15%FBS, 100 mM sodium hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine(HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ murinethymocytes/ml and dispensed into ten 96-well flat bottom tissue cultureplates (Corning, United Kingdom) at 200 μl/well. Cells were fed on days2, 4, and 6 post fusion by aspirating approximately 100 μl from eachwell with an 18 G needle (Becton Dickinson) and adding 100 μl/wellplating medium described above except containing 10 units/ml IL-6 andlacking thymocytes. On days 9 to 12, supernatants from the fusion wellswere screened by antigen capture ELISA using GST and GST-PDE10 and byECL Western analysis as described above.

[0097] A positive signal of the expected size was obtained on both lanesof the Western blot using mouse blood and monoclonal antibodies withreactivity to the yeast recombinant protein were obtained in thesubsequent fusion.

EXAMPLE 6 Analysis of PDE10 Expression by in situ Hybridization

[0098] Expression of PDE10 was examined in tissue sections by in situhybridization as described below.

[0099] Preparation of Probe

[0100] An EcoRI/PstI restriction enzyme fragment from the cDNA FB93a(corresponding to nucleotides 370 through 978 in SEQ ID NO: 22) wassubcloned into a Bluescript® vector (Stratagene, La Jolla, Calif.) togenerate an expression plasmid designated PDE10A3A. The plasmid wasdigested with EcoRI and transcribed with T3 polymerase to generate anantisense probe. A sense probe was generated by digestion the plasmidwith BamHI and transcribing with T7 polymerase. The PDE10 templates weretranscribed using a RNA Transcription kit (Stratagene, La Jolla, Calif.)in a reaction containing 5 μl of 5× transcription buffer (Stratagene),30 mM DTT (Stratagene), 0.8 mM each ATP, CTP, GTP (10 mM (Stratagene),40 U RNase Block II (Stratagene), 12.5 U T3 or T7 polymerase(Stratagene), and 300 ng linearized plasmid template, 50 μCi ³⁵S-UTP(greater than 1000 Ci/mmnol, Amersham, Arlington Heights, Ill.). Themixture was incubated at 37° C. for one hour after which the templateDNA was removed by addition of 1 μl of RNase-free DNase I (Stratagene)and incubation for 15 minutes at 37° C. The probe was hydrolyzed toapproximately 250 nucleotides in length to facilitate tissue penetrationby adding 4 μl 1 M NaHCO₃ and 6 μl 1 M Na₂CO₃ for 22 minutes at 60° C.and the reaction mixture was neutralized by addition of 25 μl of asolution containing 100 μl 3 M sodium acetate, 5 μl acetic acid (VWR,So. Plainfield, N.J.), and 395 μl dH₂O. A Quick Spin G50 RNA column(5′-3′ Inc., Boulder, Colo.) was prepared according to themanufacturer's suggested protocol. The probe was placed in the center ofthe column and the column centrifuged for four minutes at 1,000 rpm in adesk top centrifuge. The column flow-through was mixed with 50 μl dH₂O,2 μl of a 10 mg/ml tRNA solution, 10 μl 3 M sodium acetate, and 200 μl100% ethanol (VWR) and the resulting mixture was incubated at −20° C.overnight. The probe solution was microfuged for 15 minutes at 4° C.,the supernatant was removed, and the pellet was resuspended in 40 μl 1×TBE containing 1 μl of 0.1 M DTT. The probe was stored at −70 ° C. untilthe in situ hybridization assay was performed.

[0101] Preparation of Tissue Samples and In Situ Hybridization

[0102] Tissues (National Disease Research Interchange, Philadelphia, Pa.and Cooperative Human Tissue Network, Philadelphia, Pa.) were sectionedat 6 μm and placed on Superfrost Plus slides (VWR). Sections were fixedfor 20 minutes at 4° C. in 4% paraformaldehyde (Sigma, St. Louis, Mo.).The slides were rinsed in three changes of 1× calcium-, magnesium-freephosphate buffered saline (CMF-PBS), dehydrated with three successivewashes with 70% ethanol, 95% ethanol and 100% ethanol, and dried for 30minutes at room temperature. The slides were placed in 70% formamide (J.T. Baker) in 2× SSC for two minutes at 70° C., rinsed in 2× SSC at 4°C., dehydrated through 70%, 95% and 100% ethanol washes, and dried for30 minutes at room temperature.

[0103] A prehybridization step was performed by placing the slides in anairtight box containing a piece of filter paper saturated with buffercontaining 50% formamide (J. T. Baker) in 4× SSC. Each section wascovered with 100 μl of rHB2 buffer consisting of 10% dextran sulfate(Sigma), 50% formamide (J. T. Baker, Phillpsburg, N.J.), 100 mM DTT(Boehringer Mannheim, Indianapolis, Ind.), 0.3 M NaCl (Sigma), 20 mMTris, pH 7.5, 5 mM EDTA (Sigma), and 1× Denhardt's solution (Sigma) andthe slides were incubated at 42° C. for two hours. The probe, asdescribed above, was prepared by mixing 4×10⁵ cpm/tissue section with 5μl of a 10 mg/ml tRNA solution per section and heating the mixture at95° C. for three minutes. Ice cold rHB2 buffer was added to bring thefinal volume to 20 μl/section. The probe-containing solution (20μl/section) was added to 100 μl rHB2 buffer previously applied. Theslides were incubated at 55° C. for 12 to 16 hours. Followinghybridization, the slides were washed once in 4× SSC containing 10 mMDTT for one hour at room temperature, once in 50% deionized formamide(J. T. Baker), 1× SSC, and 1 mM DTT for 40 minutes at 60° C., once in 2×SSC for 30 minutes at room temperature, and once in 0.1× SSC for 30minutes at room temperature. The sections were dehydrated through 70%,95%, and 100% ethanol washes and air dried for 30 minutes. The slideswere dipped in Kodak NTB2 nuclear emulsion, dried for one to three hoursat room temperature in the dark, and stored in the dark at 4° C. withdesiccant until time of development. The slides were developed in 4° C.Kodak Dektol® developer for two minutes, dipped four times in 4° C.dH₂O, and placed in 4° C. Kodak fixer for ten minutes. The slides wererinsed in dH₂O and a standard hematoxylin and eosin (H&E) stain wasperformed as follows.

[0104] The slides were rinsed in dH2O and stained with hematoxylin andeosin by transfer of the slides through a series of the following steps:five minutes in formaldehyde/alcohol (100 ml formaldehyde, 900 ml 80%ethanol); three rinses in water for a total of two minutes; five minutesin 0.75% Harris hematoxylin (Sigma); three rinses in water for a totalof two minutes; one dip in 1% HCl/50% ethanol; one rinse in water; fourdips in 1% lithium carbonate; ten minutes in tap water; two minutes in0.5% eosin (Sigma); three rinses in water for a total of two minutes;two minutes in 70% ethanol; three one-minute rinses in 95% ethanol; twoone-minute rinses in 100% ethanol; and two two-minute rinses in xylene.Slides were mounted with cytoseal 60 (Stephens Scientific, Riverdale,N.J.).

[0105] The signals obtained with an antisense PDE10 probe were comparedto the control signals generated by a sense PDE10 probe and any signalspecific to the antisense probe was assumed to represent PDE10expression. PDE10 signal was detected throughout much of the cerebellum,with very strong signal in the Purkinje cells.

EXAMPLE 7 High Throughput Screening for PDE10 Inhibitors

[0106] In an attempt to identify specific inhibitors, PDE 10 wasscreened against a chemical library containing compounds of knownstructure. Initial screening was performed on pools of compounds (22compounds per pool) with each compound present at 4.6 μM. Pools whichinhibited PDE10 activity by greater than 50% were selected and theindividual compounds in the pool were screened at a concentration of 20μM. IC₅₀ values were determined for compounds that inhibited enzymeactivity.

[0107] An extract was prepared from Saccharomyces cerevisiae strainBJ2-54 (described in Example 4) lacking endogenous PDE activity andhaving PDE10 at an activity of 49 nmol cGMP hydrolyzed/min/ml with 32 nMcGMP. The extract was diluted 1:21,000-fold for use in the assay.Dilution buffer included 25 mM Tris, pH 8.0, 0.1 mM DTT, 5.0 mM MgCl₂,100 mM NaCl, 5 μM ZnSO₄ and 100 μg/ml BSA. PDE assay buffer (5×)contained 200 mM Tris, pH 8.0, 5 mM EGTA, 25 mM MgCl₂ and 0.5 mg/ml BSA.Just prior to screening, 5× PDE assay buffer, deionized water, and5′-nucleosidase (stock solution 15 mg/ml snake venom 5′-nucleosidase in20 mM Tris, pH 8.0) were mixed at ratios of 4:4:1 to make Assay ReagentMix.

[0108] A Packard MultiPROBE® was used to add 45 μl of the Assay ReagentMix and 20 μl of the chemical compound pools. A BIOMEK® 1000 (SeeExample 4) was used to add 20 μl of PDE10 extract diluted as describedabove and 20 μl ³²P-cGMP (ICN, specific activity 250 μCi/mmol, dilutedto 0.4 μCi/ml, 16 nM, in deionized water). Final cGMP concentration inthe assay was 0.08 μCi/ml, 3.2 nM. Ten minutes after addition of³²P-cGMP, 140 μl of 25 mg/ml charcoal (in 0.1 M NaH₂PO₄) was added tostop the reaction. After a 20 minute incubation at room temperature, theassay plates were centrifuged for five minutes at 3,500 rpm in a BeckmanGS-6R centrifuge. A BIOMEK® 1000 was used to transfer 140 μl of thesupernatant to a Wallac counting plate and Cerenkov radiation wasmeasured in a Wallac MicroBeta Counter.

[0109] Several compounds that merit further investigation were found toinhibit enzyme activity.

[0110] Numerous modifications and variations in the invention as setforth in the above illustrative examples are expected to occur to thoseskilled in the art. Consequently only such limitations as appear in theappended claims should be placed on the invention.

1 26 1 1548 DNA Homo sapiens CDS (26)..(1423) 1 cccaaggcca tctacctggacatcg atg gac gca ttc aga agc act ccg tac 52 Met Asp Ala Phe Arg Ser ThrPro Tyr 1 5 aaa gtg aga cct gtg gcc atc aag caa ctc tcc gag aga gaa gaatta 100 Lys Val Arg Pro Val Ala Ile Lys Gln Leu Ser Glu Arg Glu Glu Leu10 15 20 25 atc cag agc gtg ctg gcg cag gtt gca gag cag ttc tca aga gcattc 148 Ile Gln Ser Val Leu Ala Gln Val Ala Glu Gln Phe Ser Arg Ala Phe30 35 40 aaa atc aat gaa ctg aaa gct gaa gtt gca aat cac ttg gct gtc cta196 Lys Ile Asn Glu Leu Lys Ala Glu Val Ala Asn His Leu Ala Val Leu 4550 55 gag aaa cgc gtg gaa ttg gaa gga cta aaa gtg gtg gag att gag aaa244 Glu Lys Arg Val Glu Leu Glu Gly Leu Lys Val Val Glu Ile Glu Lys 6065 70 tgc aag agt gac att aag aag atg agg gag gag ctg gcg gcc aga agc292 Cys Lys Ser Asp Ile Lys Lys Met Arg Glu Glu Leu Ala Ala Arg Ser 7580 85 agc agg acc aac tgc ccc tgt aag tac agt ttt ttg gat aac cac aag340 Ser Arg Thr Asn Cys Pro Cys Lys Tyr Ser Phe Leu Asp Asn His Lys 9095 100 105 aag ttg act cct cga cgc gat gtt ccc act tac ccc aag tac ctgctc 388 Lys Leu Thr Pro Arg Arg Asp Val Pro Thr Tyr Pro Lys Tyr Leu Leu110 115 120 tct cca gag acc atc gag gcc ctg cgg aag ccg acc ttt gac gtctgg 436 Ser Pro Glu Thr Ile Glu Ala Leu Arg Lys Pro Thr Phe Asp Val Trp125 130 135 ctt tgg gag ccc aat gag atg ctg agc tgc ctg gag cac atg taccac 484 Leu Trp Glu Pro Asn Glu Met Leu Ser Cys Leu Glu His Met Tyr His140 145 150 gac ctc ggg ctg gtc agg gac ttc agc atc aac cct gtc acc ctcagg 532 Asp Leu Gly Leu Val Arg Asp Phe Ser Ile Asn Pro Val Thr Leu Arg155 160 165 agg tgg ctg ttc tgc gtc cac gac aac tac aga aac aac ccc ttccac 580 Arg Trp Leu Phe Cys Val His Asp Asn Tyr Arg Asn Asn Pro Phe His170 175 180 185 aac ttc cgg cac tgc ttc tgc gtg gcc cag atg atg tac agcatg gtc 628 Asn Phe Arg His Cys Phe Cys Val Ala Gln Met Met Tyr Ser MetVal 190 195 200 tgg ctc tgc agt ctc cag gag aag ttc tca caa acg gat atcctg atc 676 Trp Leu Cys Ser Leu Gln Glu Lys Phe Ser Gln Thr Asp Ile LeuIle 205 210 215 cta atg aca gcg gcc atc tgc cac gat ctg gac cat ccc ggctac aac 724 Leu Met Thr Ala Ala Ile Cys His Asp Leu Asp His Pro Gly TyrAsn 220 225 230 aac acg tac cag atc aat gcc cgc aca gag ctg gcg gtc cgctac aat 772 Asn Thr Tyr Gln Ile Asn Ala Arg Thr Glu Leu Ala Val Arg TyrAsn 235 240 245 gac atc tca ccg ctg gag aac cac cac tgc gcc gtg gcc ttccag atc 820 Asp Ile Ser Pro Leu Glu Asn His His Cys Ala Val Ala Phe GlnIle 250 255 260 265 ctc gcc gag cct gag tgc aac atc ttc tcc aac atc ccacct gat ggg 868 Leu Ala Glu Pro Glu Cys Asn Ile Phe Ser Asn Ile Pro ProAsp Gly 270 275 280 ttc aag cag atc cga cag gga atg atc aca tta atc ttggcc act gac 916 Phe Lys Gln Ile Arg Gln Gly Met Ile Thr Leu Ile Leu AlaThr Asp 285 290 295 atg gca aga cat gca gaa att atg gat tct ttc aaa gagaaa atg gag 964 Met Ala Arg His Ala Glu Ile Met Asp Ser Phe Lys Glu LysMet Glu 300 305 310 aat ttt gac tac agc aac gag gag cac atg acc ctg ctgaag atg att 1012 Asn Phe Asp Tyr Ser Asn Glu Glu His Met Thr Leu Leu LysMet Ile 315 320 325 ttg ata aaa tgc tgt gat atc tct aac gag gtc cgt ccaatg gaa gtc 1060 Leu Ile Lys Cys Cys Asp Ile Ser Asn Glu Val Arg Pro MetGlu Val 330 335 340 345 gca gag cct tgg gtg gac tgt tta tta gag gaa tatttt atg cag agc 1108 Ala Glu Pro Trp Val Asp Cys Leu Leu Glu Glu Tyr PheMet Gln Ser 350 355 360 gac cgt gag aag tca gaa ggc ctt cct gtg gca ccgttc atg gac cga 1156 Asp Arg Glu Lys Ser Glu Gly Leu Pro Val Ala Pro PheMet Asp Arg 365 370 375 gac aaa gtg acc aag gcc aca gcc cag att ggg ttcatc aag ttt gtc 1204 Asp Lys Val Thr Lys Ala Thr Ala Gln Ile Gly Phe IleLys Phe Val 380 385 390 ctg atc cca atg ttt gaa aca gtg acc aag ctc ttcccc atg gtt gag 1252 Leu Ile Pro Met Phe Glu Thr Val Thr Lys Leu Phe ProMet Val Glu 395 400 405 gag atc atg ctg cag cca ctt tgg gaa tcc cga gatcgc tac gag gag 1300 Glu Ile Met Leu Gln Pro Leu Trp Glu Ser Arg Asp ArgTyr Glu Glu 410 415 420 425 ctg aag cgg ata gat gac gcc atg aaa gag ttacag aag aag act gac 1348 Leu Lys Arg Ile Asp Asp Ala Met Lys Glu Leu GlnLys Lys Thr Asp 430 435 440 agc ttg acg tct ggg gcc acc gag aag tcc agaggg aga agc aga gat 1396 Ser Leu Thr Ser Gly Ala Thr Glu Lys Ser Arg GlyArg Ser Arg Asp 445 450 455 gtg aaa aac agt gaa gga gac tgt gcctgaggaaagc ggggggcgtg 1443 Val Lys Asn Ser Glu Gly Asp Cys Ala 460 465gctgcagttc tggacgggct ggccgagctg cgcgggatcc ttgtgcaggg aagagctgcc 1503ctgggcacct ggcaccacaa gaccatgttt tctaagaacc atttt 1548 2 466 PRT Homosapiens 2 Met Asp Ala Phe Arg Ser Thr Pro Tyr Lys Val Arg Pro Val AlaIle 1 5 10 15 Lys Gln Leu Ser Glu Arg Glu Glu Leu Ile Gln Ser Val LeuAla Gln 20 25 30 Val Ala Glu Gln Phe Ser Arg Ala Phe Lys Ile Asn Glu LeuLys Ala 35 40 45 Glu Val Ala Asn His Leu Ala Val Leu Glu Lys Arg Val GluLeu Glu 50 55 60 Gly Leu Lys Val Val Glu Ile Glu Lys Cys Lys Ser Asp IleLys Lys 65 70 75 80 Met Arg Glu Glu Leu Ala Ala Arg Ser Ser Arg Thr AsnCys Pro Cys 85 90 95 Lys Tyr Ser Phe Leu Asp Asn His Lys Lys Leu Thr ProArg Arg Asp 100 105 110 Val Pro Thr Tyr Pro Lys Tyr Leu Leu Ser Pro GluThr Ile Glu Ala 115 120 125 Leu Arg Lys Pro Thr Phe Asp Val Trp Leu TrpGlu Pro Asn Glu Met 130 135 140 Leu Ser Cys Leu Glu His Met Tyr His AspLeu Gly Leu Val Arg Asp 145 150 155 160 Phe Ser Ile Asn Pro Val Thr LeuArg Arg Trp Leu Phe Cys Val His 165 170 175 Asp Asn Tyr Arg Asn Asn ProPhe His Asn Phe Arg His Cys Phe Cys 180 185 190 Val Ala Gln Met Met TyrSer Met Val Trp Leu Cys Ser Leu Gln Glu 195 200 205 Lys Phe Ser Gln ThrAsp Ile Leu Ile Leu Met Thr Ala Ala Ile Cys 210 215 220 His Asp Leu AspHis Pro Gly Tyr Asn Asn Thr Tyr Gln Ile Asn Ala 225 230 235 240 Arg ThrGlu Leu Ala Val Arg Tyr Asn Asp Ile Ser Pro Leu Glu Asn 245 250 255 HisHis Cys Ala Val Ala Phe Gln Ile Leu Ala Glu Pro Glu Cys Asn 260 265 270Ile Phe Ser Asn Ile Pro Pro Asp Gly Phe Lys Gln Ile Arg Gln Gly 275 280285 Met Ile Thr Leu Ile Leu Ala Thr Asp Met Ala Arg His Ala Glu Ile 290295 300 Met Asp Ser Phe Lys Glu Lys Met Glu Asn Phe Asp Tyr Ser Asn Glu305 310 315 320 Glu His Met Thr Leu Leu Lys Met Ile Leu Ile Lys Cys CysAsp Ile 325 330 335 Ser Asn Glu Val Arg Pro Met Glu Val Ala Glu Pro TrpVal Asp Cys 340 345 350 Leu Leu Glu Glu Tyr Phe Met Gln Ser Asp Arg GluLys Ser Glu Gly 355 360 365 Leu Pro Val Ala Pro Phe Met Asp Arg Asp LysVal Thr Lys Ala Thr 370 375 380 Ala Gln Ile Gly Phe Ile Lys Phe Val LeuIle Pro Met Phe Glu Thr 385 390 395 400 Val Thr Lys Leu Phe Pro Met ValGlu Glu Ile Met Leu Gln Pro Leu 405 410 415 Trp Glu Ser Arg Asp Arg TyrGlu Glu Leu Lys Arg Ile Asp Asp Ala 420 425 430 Met Lys Glu Leu Gln LysLys Thr Asp Ser Leu Thr Ser Gly Ala Thr 435 440 445 Glu Lys Ser Arg GlyArg Ser Arg Asp Val Lys Asn Ser Glu Gly Asp 450 455 460 Cys Ala 465 3225 DNA Homo sapiens misc feature 130 N = A, T, G, or C 3 agcgaccgtgagaagtcaga aggccttcct gtggaaccgt tcatggaccg agacaaagtg 60 accaaggccacagcccagat tgggttcatc aagtttgccc tgatcccaat gtttgaaaca 120 gtgaccaagntcttccccat ggttgaggag atcatgctgc agccactttg ggaatcccga 180 gatcgntacgaggagctgaa gcggntagat gacgccatga aagag 225 4 158 DNA Homo sapiens miscfeature 12 N = A, T, G, or C 4 gtaccagatc antgcccgca cagagctggcggtccgntac aatgacatct caccgttgga 60 gnaaccacca ctgcgccgtg gccttccagatcctcgccga gcctgagtgn aacatcttct 120 ccaacatccc acctgatggg ttcaagcagatccgacag 158 5 98 DNA Homo sapiens misc feature 14 N = A, T, G, or C 5gagaacacca ctgngccgtg gncttccaga tcctcgccga gcctgagtgn aacatcttct 60ccaacatccc acctgatggg ttcaagcaga tccgacag 98 6 418 DNA Homo sapiens miscfeature 1 N = A, T, G, or C 6 nggttaactg gcgcatcttg tctttctctgagaacagcga tctggttatg gggcatttct 60 gtctctaatg tcactgtctg ctgcattccctgcagagcga ccgtgagaag tcagaaggcc 120 ttcccgtggc cccgttcatg gaccgagacaaagtgaccaa ggccacagcc caggattggg 180 tttcatcaag tttgtcctga tcccaatgtttgaaacagtg accaagctct tccccatggg 240 ttgagggaga ttcatgctgg cagccantttggggaatccc gaggattcgc tacgagggag 300 cttgaagcgg gattaggatg gacggccatggaaaggagtt ttacaggaag gnaggatttg 360 acagttttga agttttgggg gggccaccgaggaagttccn ggaggaggag naggcaga 418 7 428 DNA Homo sapiens misc feature 1N = A, T, G, or C 7 nagaaaaaag tgaacaaaat ggttcttaga aaacatggtcttgtggtgcc aggtgcccag 60 ggagctcttc cctgcacaag gntcccgcgc antcggccagcccgtccaga actgcagcca 120 cgccccccgn tttcctcagg cacagtctcc ttcactgtttttcacatctc tgcttctctc 180 tctggacttc tcggtggccc cagacgtcaa gctgtcagtcttcttctgta actctttcat 240 gggcgtcatc tatccgcttc agctcctcgt aggcgatctcggggattccc aaagtgggct 300 gcagcatgat cttcctcaac catggggggg aggagcttggggcactngtt ttcaaaaatt 360 gggggatcag gggacaaact ttgattggan cccatnttggggcttttggg cctttggggc 420 aatttttg 428 8 438 DNA Homo sapiens miscfeature 63 N = A, T, G, or C 8 tttttttttt ttttttttgt atcagtgaacaaaatggttc ttagaaaaca tggtcttgtg 60 gtnccaggtg cccagggagc tcttccctgcacaaggancc cgcgcantcg gccagcccgt 120 ccagaactgc agccacgccc cccgttttcctcaggcacag tctccttcac tgtttttcac 180 atctctgntt ctctctctgg ganttntcggtgggccccag aacgtcaagc tgtcagtntt 240 cttctgtaac tntttcatgg gcgtcatctatccgtttcag cttcctcgta ggcgatnttg 300 gggattccca aagtgggctg gcagcatggatcttcctcaa accatggggg gaaggagttt 360 gggtcaattn ttttcaaaac attgggggntcagggacaaa attttgatgg aaacccaatt 420 tgggggntgt gggccttg 438 9 262 DNAMus musculus 9 gagaattttg actacagcaa cgaggagcac ctgaccctgc tgaagatgattctcataaaa 60 tgctgtgata tctccaatga agtccgtccc atggaggtgg cagaatcgtgggtggactgt 120 ttactggaag aatattttat gcagagtgac cgtgagaagt ccgaagccttcctgtggccc 180 cattcatgga ccgagacaaa gtgaccaaag caacagccca aattgggttcatcaagtttg 240 tcctgatccc aatgtttgaa ac 262 10 250 DNA Mus musculus 10gagaattttg actacagcaa cgaggagcac ctgaccctgc tgaagatgat tctcataaaa 60tgctgtgata tctccaatga agtccgtccc atggaggtgg cagaatcgtg ggtggactgt 120ttactggaag aatattttat gcagagtgac cgtgagaagt ccgaagcctt cctgtggccc 180attcatggac cgagacaaag tgaccaaagc aacagccaaa ttgggttcat caagtttgtc 240tgtccaatgt 250 11 459 DNA Homo sapiens misc feature 155 N = A, T, G, orC 11 attaatcttg gccactgaca tggcaagaca tgcagaaatt atggattctt tcaaagagaa60 aatggagaat tttgactaca gcaacgagga gcacatgacc ctggtgagtg gcttattctg 120cctgggtggg cagccaggcg gttgggctgg cgaanaggtt catccatcca gctcacactg 180gaagccaaga agctgaaatt attagtcttc ttggaacaag gtgtctataa atctggtttt 240caaggtcatg actcttacta ggaaagtccg ggcagggcct ccctcctgat gggtcctcct 300tcatggtcag aggcagcatt ctcccattcc tccatctctt ttgggatttt gaaggagata 360aagtggggtg aaggccgtgc attctcgctc tgnttttcca gagaattaaa accagttttc 420ccttgaaggc acagccccag cntggcattt tgaaagttg 459 12 599 DNA Homo sapiensCDS (99)..(443) 12 tggccctcga ggccaagaat tcggcacgag tggttaactggcgcatcttg tctttctctg 60 agaacagcga tctggttatg gggcatttct gtctctaa tgtcac tgt ctg ctg cat 116 Cys His Cys Leu Leu His 1 5 tcc ctg cag agc gaccgt gag aag tca gaa ggc ctt ccc gtg gcc ccg 164 Ser Leu Gln Ser Asp ArgGlu Lys Ser Glu Gly Leu Pro Val Ala Pro 10 15 20 ttc atg gac cga gac aaagtg acc aag gcc aca gcc cag att ggg ttc 212 Phe Met Asp Arg Asp Lys ValThr Lys Ala Thr Ala Gln Ile Gly Phe 25 30 35 atc aag ttt gtc ctg atc ccaatg ttt gaa aca gtg acc aag ctc ttc 260 Ile Lys Phe Val Leu Ile Pro MetPhe Glu Thr Val Thr Lys Leu Phe 40 45 50 ccc atg gtt gag gag atc atg ctgcag cca ctt tgg gaa tcc cga gat 308 Pro Met Val Glu Glu Ile Met Leu GlnPro Leu Trp Glu Ser Arg Asp 55 60 65 70 cgc tac gag gag ctg aag cgg atagat gac gcc atg aaa gag tta cag 356 Arg Tyr Glu Glu Leu Lys Arg Ile AspAsp Ala Met Lys Glu Leu Gln 75 80 85 aag aag act gac agc ttg acg tct ggggcc acc gag aag tcc aga gag 404 Lys Lys Thr Asp Ser Leu Thr Ser Gly AlaThr Glu Lys Ser Arg Glu 90 95 100 aga agc aga gat gtg aaa aac agt gaagga gac tgt gcc tgaggaaagc 453 Arg Ser Arg Asp Val Lys Asn Ser Glu GlyAsp Cys Ala 105 110 115 ggggggcgtg gctgcagttc tggacgggct ggccgagctgcgcgggatcc ttgtgcaggg 513 aagagctgcc ctgggcacct ggcaccacaa gaccatgttttctaagaacc attttgttca 573 ctgatacaaa aaaaaaaaaa aaaaaa 599 13 115 PRTHomo sapiens 13 Cys His Cys Leu Leu His Ser Leu Gln Ser Asp Arg Glu LysSer Glu 1 5 10 15 Gly Leu Pro Val Ala Pro Phe Met Asp Arg Asp Lys ValThr Lys Ala 20 25 30 Thr Ala Gln Ile Gly Phe Ile Lys Phe Val Leu Ile ProMet Phe Glu 35 40 45 Thr Val Thr Lys Leu Phe Pro Met Val Glu Glu Ile MetLeu Gln Pro 50 55 60 Leu Trp Glu Ser Arg Asp Arg Tyr Glu Glu Leu Lys ArgIle Asp Asp 65 70 75 80 Ala Met Lys Glu Leu Gln Lys Lys Thr Asp Ser LeuThr Ser Gly Ala 85 90 95 Thr Glu Lys Ser Arg Glu Arg Ser Arg Asp Val LysAsn Ser Glu Gly 100 105 110 Asp Cys Ala 115 14 28 DNA ArtificialSequence Description of Artificial Sequence primer 14 agtcgaattcaccgtgagaa gtcagaag 28 15 28 DNA Artificial Sequence Description ofArtificial Sequence primer 15 gtcaaagctt acatggtctt gtggtgcc 28 16 1303DNA Homo sapiens CDS (107)..(1066) 16 agtgactcta ctttgtgaaa atgtgaaacttcgtgtaggt actcagtaaa tcagtaaatt 60 cttactaacg ttagccccca gcctagctatggagggtgca tgctga gcc ctg gag 115 Ala Leu Glu 1 cac atg tac cac gac ctcggg ctg gtc agg gac ttc agc atc aac cct 163 His Met Tyr His Asp Leu GlyLeu Val Arg Asp Phe Ser Ile Asn Pro 5 10 15 gtc acc ctc agg agg tgg ctgttc tgc gtc cac gac aac tac aga aac 211 Val Thr Leu Arg Arg Trp Leu PheCys Val His Asp Asn Tyr Arg Asn 20 25 30 35 aac ccc ttc cac aac ttc cggcac tgc ttc tgc gtg gcc cag atg atg 259 Asn Pro Phe His Asn Phe Arg HisCys Phe Cys Val Ala Gln Met Met 40 45 50 tac agc atg gtc tgg ctc tgc agtctc cag gag aag ttc tca caa acg 307 Tyr Ser Met Val Trp Leu Cys Ser LeuGln Glu Lys Phe Ser Gln Thr 55 60 65 gat atc ctg atc cta atg aca gcg gccatc tgc cac gat ctg gac cat 355 Asp Ile Leu Ile Leu Met Thr Ala Ala IleCys His Asp Leu Asp His 70 75 80 ccc ggc tac aac aac acg tac cag atc aatgcc cgc aca gag ctg gcg 403 Pro Gly Tyr Asn Asn Thr Tyr Gln Ile Asn AlaArg Thr Glu Leu Ala 85 90 95 gtc cgc tac aat gac atc tca ccg ctg gag aaccac cac tgc gcc gtg 451 Val Arg Tyr Asn Asp Ile Ser Pro Leu Glu Asn HisHis Cys Ala Val 100 105 110 115 gcc ttc cag atc ctc gcc gag cct gag tgcaac atc ttc tcc aac atc 499 Ala Phe Gln Ile Leu Ala Glu Pro Glu Cys AsnIle Phe Ser Asn Ile 120 125 130 cca cct gat ggg ttc aag cag atc cga caggga atg atc aca tta atc 547 Pro Pro Asp Gly Phe Lys Gln Ile Arg Gln GlyMet Ile Thr Leu Ile 135 140 145 ttg gcc act gac atg gca aga cat gca gaaatt atg gat tct ttc aaa 595 Leu Ala Thr Asp Met Ala Arg His Ala Glu IleMet Asp Ser Phe Lys 150 155 160 gag aaa atg gag aat ttt gac tac agc aacgag gag cac atg acc ctg 643 Glu Lys Met Glu Asn Phe Asp Tyr Ser Asn GluGlu His Met Thr Leu 165 170 175 ctg aag atg att ttg ata aaa tgc tgt gatatc tct aac gag gtc cgt 691 Leu Lys Met Ile Leu Ile Lys Cys Cys Asp IleSer Asn Glu Val Arg 180 185 190 195 cca atg gaa gtc gca gag cct tgg gtggac tgt tta tta gag gaa tat 739 Pro Met Glu Val Ala Glu Pro Trp Val AspCys Leu Leu Glu Glu Tyr 200 205 210 ttt atg cag agc gac cgt gag aag tcagaa ggc ctt cct gtg gca ccg 787 Phe Met Gln Ser Asp Arg Glu Lys Ser GluGly Leu Pro Val Ala Pro 215 220 225 ttc atg gac cga gac aaa gtg acc aaggcc aca gcc cag att ggg ttc 835 Phe Met Asp Arg Asp Lys Val Thr Lys AlaThr Ala Gln Ile Gly Phe 230 235 240 atc aag ttt gtc ctg atc cca atg tttgaa aca gtg acc aag ctc ttc 883 Ile Lys Phe Val Leu Ile Pro Met Phe GluThr Val Thr Lys Leu Phe 245 250 255 ccc atg gtt gag gag atc atg ctg cagcca ctt tgg gaa tcc cga gat 931 Pro Met Val Glu Glu Ile Met Leu Gln ProLeu Trp Glu Ser Arg Asp 260 265 270 275 cgc tac gag gag ctg aag cgg atagat gac gcc atg aaa gag tta cag 979 Arg Tyr Glu Glu Leu Lys Arg Ile AspAsp Ala Met Lys Glu Leu Gln 280 285 290 aag aag act gac agc ttg acg tctggg gcc acc gag aag tcc aga gag 1027 Lys Lys Thr Asp Ser Leu Thr Ser GlyAla Thr Glu Lys Ser Arg Glu 295 300 305 aga agc aga gat gtg aaa aac agtgaa gga gac tgt gcc tgaggaaagc 1076 Arg Ser Arg Asp Val Lys Asn Ser GluGly Asp Cys Ala 310 315 320 ggggggcgtg gctgcagttc tggacgggct ggccgagctgcgcgggatcc ttgtgcaggg 1136 aagagctgcc ctgggcacct ggcaccacaa gaccatgttttctaagaacc attttgttca 1196 ctgatacaaa aaaaaaaaag gaattcatga tgctgtacagaattttattt ttaaactgtc 1256 ttttaaataa tatattctta tacggaaaaa aaaaaaaaaaaaaaaaa 1303 17 320 PRT Homo sapiens 17 Ala Leu Glu His Met Tyr His AspLeu Gly Leu Val Arg Asp Phe Ser 1 5 10 15 Ile Asn Pro Val Thr Leu ArgArg Trp Leu Phe Cys Val His Asp Asn 20 25 30 Tyr Arg Asn Asn Pro Phe HisAsn Phe Arg His Cys Phe Cys Val Ala 35 40 45 Gln Met Met Tyr Ser Met ValTrp Leu Cys Ser Leu Gln Glu Lys Phe 50 55 60 Ser Gln Thr Asp Ile Leu IleLeu Met Thr Ala Ala Ile Cys His Asp 65 70 75 80 Leu Asp His Pro Gly TyrAsn Asn Thr Tyr Gln Ile Asn Ala Arg Thr 85 90 95 Glu Leu Ala Val Arg TyrAsn Asp Ile Ser Pro Leu Glu Asn His His 100 105 110 Cys Ala Val Ala PheGln Ile Leu Ala Glu Pro Glu Cys Asn Ile Phe 115 120 125 Ser Asn Ile ProPro Asp Gly Phe Lys Gln Ile Arg Gln Gly Met Ile 130 135 140 Thr Leu IleLeu Ala Thr Asp Met Ala Arg His Ala Glu Ile Met Asp 145 150 155 160 SerPhe Lys Glu Lys Met Glu Asn Phe Asp Tyr Ser Asn Glu Glu His 165 170 175Met Thr Leu Leu Lys Met Ile Leu Ile Lys Cys Cys Asp Ile Ser Asn 180 185190 Glu Val Arg Pro Met Glu Val Ala Glu Pro Trp Val Asp Cys Leu Leu 195200 205 Glu Glu Tyr Phe Met Gln Ser Asp Arg Glu Lys Ser Glu Gly Leu Pro210 215 220 Val Ala Pro Phe Met Asp Arg Asp Lys Val Thr Lys Ala Thr AlaGln 225 230 235 240 Ile Gly Phe Ile Lys Phe Val Leu Ile Pro Met Phe GluThr Val Thr 245 250 255 Lys Leu Phe Pro Met Val Glu Glu Ile Met Leu GlnPro Leu Trp Glu 260 265 270 Ser Arg Asp Arg Tyr Glu Glu Leu Lys Arg IleAsp Asp Ala Met Lys 275 280 285 Glu Leu Gln Lys Lys Thr Asp Ser Leu ThrSer Gly Ala Thr Glu Lys 290 295 300 Ser Arg Glu Arg Ser Arg Asp Val LysAsn Ser Glu Gly Asp Cys Ala 305 310 315 320 18 1887 DNA Homo sapiens CDS(74)..(1672) 18 ctcccccgcc tcccgcggcg gctggcgtcg ggaaagtaca gtaaaaagtccgagtgcagc 60 cgccgggcgc agg atg gga tcc ggc tcc tcc agc tac cgg ccc aaggcc 109 Met Gly Ser Gly Ser Ser Ser Tyr Arg Pro Lys Ala 1 5 10 atc tacctg gac atc gat gga cgc att cag aag gta atc ttc agc aag 157 Ile Tyr LeuAsp Ile Asp Gly Arg Ile Gln Lys Val Ile Phe Ser Lys 15 20 25 tac tgc aactcc agc gac atc atg gac ctg ttc tgc atc gcc acc ggc 205 Tyr Cys Asn SerSer Asp Ile Met Asp Leu Phe Cys Ile Ala Thr Gly 30 35 40 ctg cct cgg aacacg acc atc tcc ctg ctg acc acc gac gac gcc atg 253 Leu Pro Arg Asn ThrThr Ile Ser Leu Leu Thr Thr Asp Asp Ala Met 45 50 55 60 gtc tcc atc gacccc acc atg ccc gcg aat tca gaa cgc act ccg tac 301 Val Ser Ile Asp ProThr Met Pro Ala Asn Ser Glu Arg Thr Pro Tyr 65 70 75 aaa gtg aga cct gtggcc atc aag caa ctc tcc gag aga gaa gaa tta 349 Lys Val Arg Pro Val AlaIle Lys Gln Leu Ser Glu Arg Glu Glu Leu 80 85 90 atc cag agc gtg ctg gcgcag gtt gca gag cag ttc tca aga gca ttc 397 Ile Gln Ser Val Leu Ala GlnVal Ala Glu Gln Phe Ser Arg Ala Phe 95 100 105 aaa atc aat gaa ctg aaagct gaa gtt gca aat cac ttg gct gtc cta 445 Lys Ile Asn Glu Leu Lys AlaGlu Val Ala Asn His Leu Ala Val Leu 110 115 120 gag aaa cgc gtg gaa ttggaa gga cta aaa gtg gtg gag att gag aaa 493 Glu Lys Arg Val Glu Leu GluGly Leu Lys Val Val Glu Ile Glu Lys 125 130 135 140 tgc aag agt gac attaag aag atg agg gag gag ctg gcg gcc aga agc 541 Cys Lys Ser Asp Ile LysLys Met Arg Glu Glu Leu Ala Ala Arg Ser 145 150 155 agc agg acc aac tgcccc tgt aag tac agt ttt ttg gat aac cac aag 589 Ser Arg Thr Asn Cys ProCys Lys Tyr Ser Phe Leu Asp Asn His Lys 160 165 170 aag ttg act cct cgacgc gat gtt ccc act tac ccc aag tac ctg ctc 637 Lys Leu Thr Pro Arg ArgAsp Val Pro Thr Tyr Pro Lys Tyr Leu Leu 175 180 185 tct cca gag acc atcgag gcc ctg cgg aag ccg acc ttt gac gtc tgg 685 Ser Pro Glu Thr Ile GluAla Leu Arg Lys Pro Thr Phe Asp Val Trp 190 195 200 ctt tgg gag ccc aatgag atg ctg agc tgc ctg gag cac atg tac cac 733 Leu Trp Glu Pro Asn GluMet Leu Ser Cys Leu Glu His Met Tyr His 205 210 215 220 gac ctc ggg ctggtc agg gac ttc agc atc aac cct gtc acc ctc agg 781 Asp Leu Gly Leu ValArg Asp Phe Ser Ile Asn Pro Val Thr Leu Arg 225 230 235 agg tgg ctg ttctgc gtc cac gac aac tac aga aac aac ccc ttc cac 829 Arg Trp Leu Phe CysVal His Asp Asn Tyr Arg Asn Asn Pro Phe His 240 245 250 aac ttc cgg cactgc ttc tgc gtg gcc cag atg atg tac agc atg gtc 877 Asn Phe Arg His CysPhe Cys Val Ala Gln Met Met Tyr Ser Met Val 255 260 265 tgg ctc tgc agtctc cag gag aag ttc tca caa acg gat atc ctg atc 925 Trp Leu Cys Ser LeuGln Glu Lys Phe Ser Gln Thr Asp Ile Leu Ile 270 275 280 cta atg aca gcggcc atc tgc cac gat ctg gac cat ccc ggc tac aac 973 Leu Met Thr Ala AlaIle Cys His Asp Leu Asp His Pro Gly Tyr Asn 285 290 295 300 aac acg taccag atc aat gcc cgc aca gag ctg gcg gtc cgc tac aat 1021 Asn Thr Tyr GlnIle Asn Ala Arg Thr Glu Leu Ala Val Arg Tyr Asn 305 310 315 gac atc tcaccg ctg gag aac cac cac tgc gcc gtg gcc ttc cag atc 1069 Asp Ile Ser ProLeu Glu Asn His His Cys Ala Val Ala Phe Gln Ile 320 325 330 ctc gcc gagcct gag tgc aac atc ttc tcc aac atc cca cct gat ggg 1117 Leu Ala Glu ProGlu Cys Asn Ile Phe Ser Asn Ile Pro Pro Asp Gly 335 340 345 ttc aag cagatc cga cag gga atg atc aca tta atc ttg gcc act gac 1165 Phe Lys Gln IleArg Gln Gly Met Ile Thr Leu Ile Leu Ala Thr Asp 350 355 360 atg gca agacat gca gaa att atg gat tct ttc aaa gag aaa atg gag 1213 Met Ala Arg HisAla Glu Ile Met Asp Ser Phe Lys Glu Lys Met Glu 365 370 375 380 aat tttgac tac agc aac gag gag cac atg acc ctg ctg aag atg att 1261 Asn Phe AspTyr Ser Asn Glu Glu His Met Thr Leu Leu Lys Met Ile 385 390 395 ttg ataaaa tgc tgt gat atc tct aac gag gtc cgt cca atg gaa gtc 1309 Leu Ile LysCys Cys Asp Ile Ser Asn Glu Val Arg Pro Met Glu Val 400 405 410 gca gagcct tgg gtg gac tgt tta tta gag gaa tat ttt atg cag agc 1357 Ala Glu ProTrp Val Asp Cys Leu Leu Glu Glu Tyr Phe Met Gln Ser 415 420 425 gac cgtgag aag tca gaa ggc ctt cct gtg gca ccg ttc atg gac cga 1405 Asp Arg GluLys Ser Glu Gly Leu Pro Val Ala Pro Phe Met Asp Arg 430 435 440 gac aaagtg acc aag gcc aca gcc cag att ggg ttc atc aag ttt gtc 1453 Asp Lys ValThr Lys Ala Thr Ala Gln Ile Gly Phe Ile Lys Phe Val 445 450 455 460 ctgatc cca atg ttt gaa aca gtg acc aag ctc ttc ccc atg gtt gag 1501 Leu IlePro Met Phe Glu Thr Val Thr Lys Leu Phe Pro Met Val Glu 465 470 475 gagatc atg ctg cag cca ctt tgg gaa tcc cga gat cgc tac gag gag 1549 Glu IleMet Leu Gln Pro Leu Trp Glu Ser Arg Asp Arg Tyr Glu Glu 480 485 490 ctgaag cgg ata gat gac gcc atg aaa gag tta cag aag aag act gac 1597 Leu LysArg Ile Asp Asp Ala Met Lys Glu Leu Gln Lys Lys Thr Asp 495 500 505 agcttg acg tct ggg gcc acc gag aag tcc aga gag aga agc aga gat 1645 Ser LeuThr Ser Gly Ala Thr Glu Lys Ser Arg Glu Arg Ser Arg Asp 510 515 520 gtgaaa aac agt gaa gga gac tgt gcc tgaggaaagc ggggggcgtg 1692 Val Lys AsnSer Glu Gly Asp Cys Ala 525 530 gctgcagttc tggacgggct ggccgagctgcgcgggatcc ttgtgcaggg aagagctgcc 1752 ctgggcacct ggcaccacaa gaccatgttttctaagaacc attttgttca ctgataaaaa 1812 aaaaaaaaaa ggaattcatg atgctgtacagaattttatt tttaaactgt cttttaaata 1872 atatattctt atacg 1887 19 533 PRTHomo sapiens 19 Met Gly Ser Gly Ser Ser Ser Tyr Arg Pro Lys Ala Ile TyrLeu Asp 1 5 10 15 Ile Asp Gly Arg Ile Gln Lys Val Ile Phe Ser Lys TyrCys Asn Ser 20 25 30 Ser Asp Ile Met Asp Leu Phe Cys Ile Ala Thr Gly LeuPro Arg Asn 35 40 45 Thr Thr Ile Ser Leu Leu Thr Thr Asp Asp Ala Met ValSer Ile Asp 50 55 60 Pro Thr Met Pro Ala Asn Ser Glu Arg Thr Pro Tyr LysVal Arg Pro 65 70 75 80 Val Ala Ile Lys Gln Leu Ser Glu Arg Glu Glu LeuIle Gln Ser Val 85 90 95 Leu Ala Gln Val Ala Glu Gln Phe Ser Arg Ala PheLys Ile Asn Glu 100 105 110 Leu Lys Ala Glu Val Ala Asn His Leu Ala ValLeu Glu Lys Arg Val 115 120 125 Glu Leu Glu Gly Leu Lys Val Val Glu IleGlu Lys Cys Lys Ser Asp 130 135 140 Ile Lys Lys Met Arg Glu Glu Leu AlaAla Arg Ser Ser Arg Thr Asn 145 150 155 160 Cys Pro Cys Lys Tyr Ser PheLeu Asp Asn His Lys Lys Leu Thr Pro 165 170 175 Arg Arg Asp Val Pro ThrTyr Pro Lys Tyr Leu Leu Ser Pro Glu Thr 180 185 190 Ile Glu Ala Leu ArgLys Pro Thr Phe Asp Val Trp Leu Trp Glu Pro 195 200 205 Asn Glu Met LeuSer Cys Leu Glu His Met Tyr His Asp Leu Gly Leu 210 215 220 Val Arg AspPhe Ser Ile Asn Pro Val Thr Leu Arg Arg Trp Leu Phe 225 230 235 240 CysVal His Asp Asn Tyr Arg Asn Asn Pro Phe His Asn Phe Arg His 245 250 255Cys Phe Cys Val Ala Gln Met Met Tyr Ser Met Val Trp Leu Cys Ser 260 265270 Leu Gln Glu Lys Phe Ser Gln Thr Asp Ile Leu Ile Leu Met Thr Ala 275280 285 Ala Ile Cys His Asp Leu Asp His Pro Gly Tyr Asn Asn Thr Tyr Gln290 295 300 Ile Asn Ala Arg Thr Glu Leu Ala Val Arg Tyr Asn Asp Ile SerPro 305 310 315 320 Leu Glu Asn His His Cys Ala Val Ala Phe Gln Ile LeuAla Glu Pro 325 330 335 Glu Cys Asn Ile Phe Ser Asn Ile Pro Pro Asp GlyPhe Lys Gln Ile 340 345 350 Arg Gln Gly Met Ile Thr Leu Ile Leu Ala ThrAsp Met Ala Arg His 355 360 365 Ala Glu Ile Met Asp Ser Phe Lys Glu LysMet Glu Asn Phe Asp Tyr 370 375 380 Ser Asn Glu Glu His Met Thr Leu LeuLys Met Ile Leu Ile Lys Cys 385 390 395 400 Cys Asp Ile Ser Asn Glu ValArg Pro Met Glu Val Ala Glu Pro Trp 405 410 415 Val Asp Cys Leu Leu GluGlu Tyr Phe Met Gln Ser Asp Arg Glu Lys 420 425 430 Ser Glu Gly Leu ProVal Ala Pro Phe Met Asp Arg Asp Lys Val Thr 435 440 445 Lys Ala Thr AlaGln Ile Gly Phe Ile Lys Phe Val Leu Ile Pro Met 450 455 460 Phe Glu ThrVal Thr Lys Leu Phe Pro Met Val Glu Glu Ile Met Leu 465 470 475 480 GlnPro Leu Trp Glu Ser Arg Asp Arg Tyr Glu Glu Leu Lys Arg Ile 485 490 495Asp Asp Ala Met Lys Glu Leu Gln Lys Lys Thr Asp Ser Leu Thr Ser 500 505510 Gly Ala Thr Glu Lys Ser Arg Glu Arg Ser Arg Asp Val Lys Asn Ser 515520 525 Glu Gly Asp Cys Ala 530 20 1967 DNA Homo sapiens CDS (2)..(1741)20 c tac ctg gac atc gat gga cgc att cag aag gta atc ttc agc aag tac 49Tyr Leu Asp Ile Asp Gly Arg Ile Gln Lys Val Ile Phe Ser Lys Tyr 1 5 1015 tgc aac tcc agc gac atc atg gac ctg ttc tgc atc gcc acc ggc ctg 97Cys Asn Ser Ser Asp Ile Met Asp Leu Phe Cys Ile Ala Thr Gly Leu 20 25 30cct cgg aac acg acc atc tcc ctg ctg acc acc gac gac gcc atg gtc 145 ProArg Asn Thr Thr Ile Ser Leu Leu Thr Thr Asp Asp Ala Met Val 35 40 45 tccatc gac ccc acc atg ccc gcg aat tca gaa cgc act ccg tac aaa 193 Ser IleAsp Pro Thr Met Pro Ala Asn Ser Glu Arg Thr Pro Tyr Lys 50 55 60 gtg agacct gtg gcc atc aag caa ctc tcc gct gat gtc gag gac aag 241 Val Arg ProVal Ala Ile Lys Gln Leu Ser Ala Asp Val Glu Asp Lys 65 70 75 80 aga accaca agc cgt ggc cag tct gct gag aga cca ctg agg gac aga 289 Arg Thr ThrSer Arg Gly Gln Ser Ala Glu Arg Pro Leu Arg Asp Arg 85 90 95 cgg gtt gtgggc ctg gag cag ccc cgg agg gaa gga gca ttt gaa agt 337 Arg Val Val GlyLeu Glu Gln Pro Arg Arg Glu Gly Ala Phe Glu Ser 100 105 110 gga cag gtagag ccc agg ccc aga gag ccc cag ggc tgc tac cag gaa 385 Gly Gln Val GluPro Arg Pro Arg Glu Pro Gln Gly Cys Tyr Gln Glu 115 120 125 ggc cag cgcatc cct cca gag aga gaa gaa tta atc cag agc gtg ctg 433 Gly Gln Arg IlePro Pro Glu Arg Glu Glu Leu Ile Gln Ser Val Leu 130 135 140 gcg cag gttgca gag cag ttc tca aga gca ttc aaa atc aat gaa ctg 481 Ala Gln Val AlaGlu Gln Phe Ser Arg Ala Phe Lys Ile Asn Glu Leu 145 150 155 160 aaa gctgaa gtt gca aat cac ttg gct gtc cta gag aaa cgc gtg gaa 529 Lys Ala GluVal Ala Asn His Leu Ala Val Leu Glu Lys Arg Val Glu 165 170 175 ttg gaagga cta aaa gtg gtg gag att gag aaa tgc aag agt gac att 577 Leu Glu GlyLeu Lys Val Val Glu Ile Glu Lys Cys Lys Ser Asp Ile 180 185 190 aag aagatg agg gag gag ctg gcg gcc aga agc agc agg acc aac tgc 625 Lys Lys MetArg Glu Glu Leu Ala Ala Arg Ser Ser Arg Thr Asn Cys 195 200 205 ccc tgtaag tac agt ttt ttg gat aac cac aag aag ttg act cct cga 673 Pro Cys LysTyr Ser Phe Leu Asp Asn His Lys Lys Leu Thr Pro Arg 210 215 220 cgc gatgtt ccc act tac ccc aag tac ctg ctc tct cca gag acc atc 721 Arg Asp ValPro Thr Tyr Pro Lys Tyr Leu Leu Ser Pro Glu Thr Ile 225 230 235 240 gaggcc ctg cgg aag ccg acc ttt gac gtc tgg ctt tgg gag ccc aat 769 Glu AlaLeu Arg Lys Pro Thr Phe Asp Val Trp Leu Trp Glu Pro Asn 245 250 255 gagatg ctg agc tgc ctg gag cac atg tac cac gac ctc ggg ctg gtc 817 Glu MetLeu Ser Cys Leu Glu His Met Tyr His Asp Leu Gly Leu Val 260 265 270 agggac ttc agc atc aac cct gtc acc ctc agg agg tgg ctg ttc tgc 865 Arg AspPhe Ser Ile Asn Pro Val Thr Leu Arg Arg Trp Leu Phe Cys 275 280 285 gtccac gac aac tac aga aac aac ccc ttc cac aac ttc cgg cac tgc 913 Val HisAsp Asn Tyr Arg Asn Asn Pro Phe His Asn Phe Arg His Cys 290 295 300 ttctgc gtg gcc cag atg atg tac agc atg gtc tgg ctc tgc agt ctc 961 Phe CysVal Ala Gln Met Met Tyr Ser Met Val Trp Leu Cys Ser Leu 305 310 315 320cag gag aag ttc tca caa acg gat atc ctg atc cta atg aca gcg gcc 1009 GlnGlu Lys Phe Ser Gln Thr Asp Ile Leu Ile Leu Met Thr Ala Ala 325 330 335atc tgc cac gat ctg gac cat ccc ggc tac aac aac acg tac cag atc 1057 IleCys His Asp Leu Asp His Pro Gly Tyr Asn Asn Thr Tyr Gln Ile 340 345 350aat gcc cgc aca gag ctg gcg gtc cgc tac aat gac atc tca ccg ctg 1105 AsnAla Arg Thr Glu Leu Ala Val Arg Tyr Asn Asp Ile Ser Pro Leu 355 360 365gag aac cac cac tgc gcc gtg gcc ttc cag atc ctc gcc gag cct gag 1153 GluAsn His His Cys Ala Val Ala Phe Gln Ile Leu Ala Glu Pro Glu 370 375 380tgc aac atc ttc tcc aac atc cca cct gat ggg ttc aag cag atc cga 1201 CysAsn Ile Phe Ser Asn Ile Pro Pro Asp Gly Phe Lys Gln Ile Arg 385 390 395400 cag gga atg atc aca tta atc ttg gcc act gac atg gca aga cat gca 1249Gln Gly Met Ile Thr Leu Ile Leu Ala Thr Asp Met Ala Arg His Ala 405 410415 gaa att atg gat tct ttc aaa gag aaa atg gag aat ttt gac tac agc 1297Glu Ile Met Asp Ser Phe Lys Glu Lys Met Glu Asn Phe Asp Tyr Ser 420 425430 aac gag gag cac atg acc ctg ctg aag atg att ttg ata aaa tgc tgt 1345Asn Glu Glu His Met Thr Leu Leu Lys Met Ile Leu Ile Lys Cys Cys 435 440445 gat atc tct aac gag gtc cgt cca atg gaa gtc gca gag cct tgg gtg 1393Asp Ile Ser Asn Glu Val Arg Pro Met Glu Val Ala Glu Pro Trp Val 450 455460 gac tgt tta tta gag gaa tat ttt atg cag agc gac cgt gag aag tca 1441Asp Cys Leu Leu Glu Glu Tyr Phe Met Gln Ser Asp Arg Glu Lys Ser 465 470475 480 gaa ggc ctt cct gtg gca ccg ttc atg gac cga gac aaa gtg acc aag1489 Glu Gly Leu Pro Val Ala Pro Phe Met Asp Arg Asp Lys Val Thr Lys 485490 495 gcc aca gcc cag att ggg ttc atc aag ttt gtc ctg atc cca atg ttt1537 Ala Thr Ala Gln Ile Gly Phe Ile Lys Phe Val Leu Ile Pro Met Phe 500505 510 gaa aca gtg acc aag ctc ttc ccc atg gtt gag gag atc atg ctg cag1585 Glu Thr Val Thr Lys Leu Phe Pro Met Val Glu Glu Ile Met Leu Gln 515520 525 cca ctt tgg gaa tcc cga gat cgc tac gag gag ctg aag cgg ata gat1633 Pro Leu Trp Glu Ser Arg Asp Arg Tyr Glu Glu Leu Lys Arg Ile Asp 530535 540 gac gcc atg aaa gag tta cag aag aag act gac agc ttg acg tct ggg1681 Asp Ala Met Lys Glu Leu Gln Lys Lys Thr Asp Ser Leu Thr Ser Gly 545550 555 560 gcc acc gag aag tcc aga gag aga agc aga gat gtg aaa aac agtgaa 1729 Ala Thr Glu Lys Ser Arg Glu Arg Ser Arg Asp Val Lys Asn Ser Glu565 570 575 gga gac tgt gcc tgaggaaagc ggggggcgtg gctgcagttc tggacgggct1781 Gly Asp Cys Ala 580 ggccgagctg cgcgggatcc ttgtgcaggg aagagctgccctgggcacct ggcaccacaa 1841 gaccatgttt tctaagaacc attttgttca ctgatacaaaaaaaaaaaaa ggaattcatg 1901 atgctgtaca gaattttatt tttaaactgt cttttaaataatatattctt atacggaaaa 1961 aaaaaa 1967 21 580 PRT Homo sapiens 21 TyrLeu Asp Ile Asp Gly Arg Ile Gln Lys Val Ile Phe Ser Lys Tyr 1 5 10 15Cys Asn Ser Ser Asp Ile Met Asp Leu Phe Cys Ile Ala Thr Gly Leu 20 25 30Pro Arg Asn Thr Thr Ile Ser Leu Leu Thr Thr Asp Asp Ala Met Val 35 40 45Ser Ile Asp Pro Thr Met Pro Ala Asn Ser Glu Arg Thr Pro Tyr Lys 50 55 60Val Arg Pro Val Ala Ile Lys Gln Leu Ser Ala Asp Val Glu Asp Lys 65 70 7580 Arg Thr Thr Ser Arg Gly Gln Ser Ala Glu Arg Pro Leu Arg Asp Arg 85 9095 Arg Val Val Gly Leu Glu Gln Pro Arg Arg Glu Gly Ala Phe Glu Ser 100105 110 Gly Gln Val Glu Pro Arg Pro Arg Glu Pro Gln Gly Cys Tyr Gln Glu115 120 125 Gly Gln Arg Ile Pro Pro Glu Arg Glu Glu Leu Ile Gln Ser ValLeu 130 135 140 Ala Gln Val Ala Glu Gln Phe Ser Arg Ala Phe Lys Ile AsnGlu Leu 145 150 155 160 Lys Ala Glu Val Ala Asn His Leu Ala Val Leu GluLys Arg Val Glu 165 170 175 Leu Glu Gly Leu Lys Val Val Glu Ile Glu LysCys Lys Ser Asp Ile 180 185 190 Lys Lys Met Arg Glu Glu Leu Ala Ala ArgSer Ser Arg Thr Asn Cys 195 200 205 Pro Cys Lys Tyr Ser Phe Leu Asp AsnHis Lys Lys Leu Thr Pro Arg 210 215 220 Arg Asp Val Pro Thr Tyr Pro LysTyr Leu Leu Ser Pro Glu Thr Ile 225 230 235 240 Glu Ala Leu Arg Lys ProThr Phe Asp Val Trp Leu Trp Glu Pro Asn 245 250 255 Glu Met Leu Ser CysLeu Glu His Met Tyr His Asp Leu Gly Leu Val 260 265 270 Arg Asp Phe SerIle Asn Pro Val Thr Leu Arg Arg Trp Leu Phe Cys 275 280 285 Val His AspAsn Tyr Arg Asn Asn Pro Phe His Asn Phe Arg His Cys 290 295 300 Phe CysVal Ala Gln Met Met Tyr Ser Met Val Trp Leu Cys Ser Leu 305 310 315 320Gln Glu Lys Phe Ser Gln Thr Asp Ile Leu Ile Leu Met Thr Ala Ala 325 330335 Ile Cys His Asp Leu Asp His Pro Gly Tyr Asn Asn Thr Tyr Gln Ile 340345 350 Asn Ala Arg Thr Glu Leu Ala Val Arg Tyr Asn Asp Ile Ser Pro Leu355 360 365 Glu Asn His His Cys Ala Val Ala Phe Gln Ile Leu Ala Glu ProGlu 370 375 380 Cys Asn Ile Phe Ser Asn Ile Pro Pro Asp Gly Phe Lys GlnIle Arg 385 390 395 400 Gln Gly Met Ile Thr Leu Ile Leu Ala Thr Asp MetAla Arg His Ala 405 410 415 Glu Ile Met Asp Ser Phe Lys Glu Lys Met GluAsn Phe Asp Tyr Ser 420 425 430 Asn Glu Glu His Met Thr Leu Leu Lys MetIle Leu Ile Lys Cys Cys 435 440 445 Asp Ile Ser Asn Glu Val Arg Pro MetGlu Val Ala Glu Pro Trp Val 450 455 460 Asp Cys Leu Leu Glu Glu Tyr PheMet Gln Ser Asp Arg Glu Lys Ser 465 470 475 480 Glu Gly Leu Pro Val AlaPro Phe Met Asp Arg Asp Lys Val Thr Lys 485 490 495 Ala Thr Ala Gln IleGly Phe Ile Lys Phe Val Leu Ile Pro Met Phe 500 505 510 Glu Thr Val ThrLys Leu Phe Pro Met Val Glu Glu Ile Met Leu Gln 515 520 525 Pro Leu TrpGlu Ser Arg Asp Arg Tyr Glu Glu Leu Lys Arg Ile Asp 530 535 540 Asp AlaMet Lys Glu Leu Gln Lys Lys Thr Asp Ser Leu Thr Ser Gly 545 550 555 560Ala Thr Glu Lys Ser Arg Glu Arg Ser Arg Asp Val Lys Asn Ser Glu 565 570575 Gly Asp Cys Ala 580 22 1457 DNA Homo sapiens CDS (164)..(1453) 22ggctcccggg cgtcccgggc ccggtggcgg cgcggctgtg gttggctgag cgccgcgggc 60cgccccccgc ccgccccctc ccctgctccc ctcccccgcc tcccgcggcg gctggcgtcg 120ggaaagtaca gtaaaaagtc cgagtgcagc cgccgggcgc agg atg gga tcc ggc 175 MetGly Ser Gly 1 tcc tcc agc tac cgg ccc aag gcc atc tac ctg gac atc gatgga cgc 223 Ser Ser Ser Tyr Arg Pro Lys Ala Ile Tyr Leu Asp Ile Asp GlyArg 5 10 15 20 att cag aag gta atc ttc agc aag tac tgc aac tcc agc gacatc atg 271 Ile Gln Lys Val Ile Phe Ser Lys Tyr Cys Asn Ser Ser Asp IleMet 25 30 35 gac ctg ttc tgc atc gcc acc ggc ctg cct cgg aac acg acc atctcc 319 Asp Leu Phe Cys Ile Ala Thr Gly Leu Pro Arg Asn Thr Thr Ile Ser40 45 50 ctg ctg acc acc gac gac gcc atg gtc tcc atc gac ccc acc atg ccc367 Leu Leu Thr Thr Asp Asp Ala Met Val Ser Ile Asp Pro Thr Met Pro 5560 65 gcg aat tca gaa cgc act ccg tac aaa gtg aga cct gtg gcc atc aag415 Ala Asn Ser Glu Arg Thr Pro Tyr Lys Val Arg Pro Val Ala Ile Lys 7075 80 caa ctc tcc gag aga gaa gaa tta atc cag agc gtg ctg gcg cag gtt463 Gln Leu Ser Glu Arg Glu Glu Leu Ile Gln Ser Val Leu Ala Gln Val 8590 95 100 gca gag cag ttc tca aga gca ttc aaa atc aat gaa ctg aaa gctgaa 511 Ala Glu Gln Phe Ser Arg Ala Phe Lys Ile Asn Glu Leu Lys Ala Glu105 110 115 gtt gca aat cac ttg gct gtc cta gag aaa cgc gtg gaa ttg gaagga 559 Val Ala Asn His Leu Ala Val Leu Glu Lys Arg Val Glu Leu Glu Gly120 125 130 cta aaa gtg gtg gag att gag aaa tgc aag agt gac att aag aagatg 607 Leu Lys Val Val Glu Ile Glu Lys Cys Lys Ser Asp Ile Lys Lys Met135 140 145 agg gag gag ctg gcg gcc aga agc agc agg acc aac tgc ccc tgtaag 655 Arg Glu Glu Leu Ala Ala Arg Ser Ser Arg Thr Asn Cys Pro Cys Lys150 155 160 tac agt ttt ttg gat aac cac aag aag ttg act cct cga cgc gatgtt 703 Tyr Ser Phe Leu Asp Asn His Lys Lys Leu Thr Pro Arg Arg Asp Val165 170 175 180 ccc act tac ccc aag tac ctg ctc tct cca gag acc atc gaggcc ctg 751 Pro Thr Tyr Pro Lys Tyr Leu Leu Ser Pro Glu Thr Ile Glu AlaLeu 185 190 195 cgg aag ccg acc ttt gac gtc tgg ctt tgg gag ccc aat gagatg ctg 799 Arg Lys Pro Thr Phe Asp Val Trp Leu Trp Glu Pro Asn Glu MetLeu 200 205 210 agc tgc ctg gag cac atg tac cac gac ctc ggg ctg gtc agggac ttc 847 Ser Cys Leu Glu His Met Tyr His Asp Leu Gly Leu Val Arg AspPhe 215 220 225 agc atc aac cct gtc acc ctc agg agg tgg ctg ttc tgc gtccac gac 895 Ser Ile Asn Pro Val Thr Leu Arg Arg Trp Leu Phe Cys Val HisAsp 230 235 240 aac tac aga aac aac ccc ttc cac aac ttc cgg cac tgc ttctgc gtg 943 Asn Tyr Arg Asn Asn Pro Phe His Asn Phe Arg His Cys Phe CysVal 245 250 255 260 gcc cag atg atg tac agc atg gtc tgg ctc tgc agt ctccag gag aag 991 Ala Gln Met Met Tyr Ser Met Val Trp Leu Cys Ser Leu GlnGlu Lys 265 270 275 ttc tca caa acg gat atc ctg atc cta atg aca gcg gccatc tgc cac 1039 Phe Ser Gln Thr Asp Ile Leu Ile Leu Met Thr Ala Ala IleCys His 280 285 290 gat ctg gac cat ccc ggc tac aac aac acg tac cag atcaat gcc cgc 1087 Asp Leu Asp His Pro Gly Tyr Asn Asn Thr Tyr Gln Ile AsnAla Arg 295 300 305 aca gag ctg gcg gtc cgc tac aat gac atc tca ccg ctggag aac cac 1135 Thr Glu Leu Ala Val Arg Tyr Asn Asp Ile Ser Pro Leu GluAsn His 310 315 320 cac tgc gcc gtg gcc ttc cag atc ctc gcc gag cct gagtgc aac atc 1183 His Cys Ala Val Ala Phe Gln Ile Leu Ala Glu Pro Glu CysAsn Ile 325 330 335 340 ttc tcc aac atc cca cct gat ggg ttc aag cag atccga cag gga atg 1231 Phe Ser Asn Ile Pro Pro Asp Gly Phe Lys Gln Ile ArgGln Gly Met 345 350 355 atc aca tta atc ttg gcc act gac atg gca aga catgca gaa att atg 1279 Ile Thr Leu Ile Leu Ala Thr Asp Met Ala Arg His AlaGlu Ile Met 360 365 370 gat tct ttc aaa gag aaa atg gag aat ttt gac tacagc aac gag gag 1327 Asp Ser Phe Lys Glu Lys Met Glu Asn Phe Asp Tyr SerAsn Glu Glu 375 380 385 cac atg acc ctg ctg aag atg att ttg ata aaa tgctgt gat atc tct 1375 His Met Thr Leu Leu Lys Met Ile Leu Ile Lys Cys CysAsp Ile Ser 390 395 400 aac gag gtc cgt cca atg gaa gtc gca gag cct tgggtg gac tgt tta 1423 Asn Glu Val Arg Pro Met Glu Val Ala Glu Pro Trp ValAsp Cys Leu 405 410 415 420 tta gag gaa tat ttt atg cag agc gac cgt gaga1457 Leu Glu Glu Tyr Phe Met Gln Ser Asp Arg 425 430 23 430 PRT Homosapiens 23 Met Gly Ser Gly Ser Ser Ser Tyr Arg Pro Lys Ala Ile Tyr LeuAsp 1 5 10 15 Ile Asp Gly Arg Ile Gln Lys Val Ile Phe Ser Lys Tyr CysAsn Ser 20 25 30 Ser Asp Ile Met Asp Leu Phe Cys Ile Ala Thr Gly Leu ProArg Asn 35 40 45 Thr Thr Ile Ser Leu Leu Thr Thr Asp Asp Ala Met Val SerIle Asp 50 55 60 Pro Thr Met Pro Ala Asn Ser Glu Arg Thr Pro Tyr Lys ValArg Pro 65 70 75 80 Val Ala Ile Lys Gln Leu Ser Glu Arg Glu Glu Leu IleGln Ser Val 85 90 95 Leu Ala Gln Val Ala Glu Gln Phe Ser Arg Ala Phe LysIle Asn Glu 100 105 110 Leu Lys Ala Glu Val Ala Asn His Leu Ala Val LeuGlu Lys Arg Val 115 120 125 Glu Leu Glu Gly Leu Lys Val Val Glu Ile GluLys Cys Lys Ser Asp 130 135 140 Ile Lys Lys Met Arg Glu Glu Leu Ala AlaArg Ser Ser Arg Thr Asn 145 150 155 160 Cys Pro Cys Lys Tyr Ser Phe LeuAsp Asn His Lys Lys Leu Thr Pro 165 170 175 Arg Arg Asp Val Pro Thr TyrPro Lys Tyr Leu Leu Ser Pro Glu Thr 180 185 190 Ile Glu Ala Leu Arg LysPro Thr Phe Asp Val Trp Leu Trp Glu Pro 195 200 205 Asn Glu Met Leu SerCys Leu Glu His Met Tyr His Asp Leu Gly Leu 210 215 220 Val Arg Asp PheSer Ile Asn Pro Val Thr Leu Arg Arg Trp Leu Phe 225 230 235 240 Cys ValHis Asp Asn Tyr Arg Asn Asn Pro Phe His Asn Phe Arg His 245 250 255 CysPhe Cys Val Ala Gln Met Met Tyr Ser Met Val Trp Leu Cys Ser 260 265 270Leu Gln Glu Lys Phe Ser Gln Thr Asp Ile Leu Ile Leu Met Thr Ala 275 280285 Ala Ile Cys His Asp Leu Asp His Pro Gly Tyr Asn Asn Thr Tyr Gln 290295 300 Ile Asn Ala Arg Thr Glu Leu Ala Val Arg Tyr Asn Asp Ile Ser Pro305 310 315 320 Leu Glu Asn His His Cys Ala Val Ala Phe Gln Ile Leu AlaGlu Pro 325 330 335 Glu Cys Asn Ile Phe Ser Asn Ile Pro Pro Asp Gly PheLys Gln Ile 340 345 350 Arg Gln Gly Met Ile Thr Leu Ile Leu Ala Thr AspMet Ala Arg His 355 360 365 Ala Glu Ile Met Asp Ser Phe Lys Glu Lys MetGlu Asn Phe Asp Tyr 370 375 380 Ser Asn Glu Glu His Met Thr Leu Leu LysMet Ile Leu Ile Lys Cys 385 390 395 400 Cys Asp Ile Ser Asn Glu Val ArgPro Met Glu Val Ala Glu Pro Trp 405 410 415 Val Asp Cys Leu Leu Glu GluTyr Phe Met Gln Ser Asp Arg 420 425 430 24 8 PRT Artificial SequenceDescription of Artificial Sequence FLAG epitope 24 Asp Thr Lys Asp AspAsp Asp Lys 1 5 25 54 DNA Artificial Sequence Description of ArtificialSequence primer 25 tagaccatgg actacaagga cgacgatgac aagatggacgcattcagaag cact 54 26 18 DNA Artificial Sequence Description ofArtificial Sequence primer 26 cgaggagtca acttcttg 18

What is claimed is:
 1. A purified and isolated PDE10 polypeptide.
 2. Thepolypeptide according to claim 1 comprising the amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 18, SEQID NO: 20 and SEQ ID NO:
 22. 3. A polynucleotide encoding thepolypeptide according to claim 1 or
 2. 4. The polynucleotide accordingto claim 3 comprising the sequence set forth in SEQ ID NO:
 1. 5. Apolynucleotide encoding a human PDE10 polypeptide selected from thegroup consisting of: a) the polynucleotide according to claim 4; b) aDNA which hybridizes under moderately stringent conditions to thenon-coding strand of the polynucleotide of (a); and c) a DNA which wouldhybridize to the non-coding strand of the polynucleotide of (a) but forthe redundancy of the genetic code.
 6. The polynucleotide of claim 5comprising the polynucleotide sequence set out in SEQ ID NO:
 18. 7. Thepolynucleotide of claim 5 comprising the polynucleotide sequence set outin SEQ ID NO:
 20. 8. The polynucleotide of claim 5 comprising thepolynucleotide sequence set out in SEQ ID NO:
 22. 9. The polynucleotideof claim 5 which is a DNA molecule.
 10. The DNA of claim 9 which is acDNA molecule.
 11. The DNA of claim 9 which is a wholly or partiallychemically synthesized DNA molecule.
 12. A polynucleotide comprising thesequence set out in SEQ ID NO: 1 or a fragment thereof.
 13. Apolynucleotide comprising the sequence set out in SEQ ID NO: 18 or afragment thereof.
 14. A polynucleotide comprising the sequence set outin SEQ ID NO: 20 or a fragment thereof.
 15. A polynucleotide comprisingthe sequence set out in SEQ ID NO: 22 or a fragment thereof.
 16. Ananti-sense polynucleotide which specifically hybridizes with thecomplement of the polynucleotide of claim
 5. 17. A expression constructcomprising the polynucleotide according to claim
 5. 18. A host celltransformed or transfected with the expression construct according toclaim
 17. 19. A method for producing a PDE10 polypeptide comprising thesteps of: a) growing the host cell according to claim 18 underconditions appropriate for expression of the PDE10 polypeptide and b)isolating the PDE10 polypeptide from the host cell or the medium of itsgrowth.
 20. An antibody specifically immunoreactive with the polypeptideaccording to claim 1 or
 2. 21. The antibody according to claim 20 whichis a monoclonal antibody.
 22. A hybridoma which produces the antibodyaccording to claim
 21. 23. An anti-idiotype antibody specificallyimmunoreactive with the antibody according to claim
 21. 24. A method toidentify a specific binding partner compound of the PDE10 polypeptideaccording to claim 1 or 2 comprising the steps of: a) contacting thePDE10 polypeptide with a compound under conditions which permit bindingbetween the compound and the PDE10 polypeptide; b) detecting binding ofthe compound to the PDE10 polypeptide; and c) identifying the compoundas a specific binding partner of the PDE10 polypeptide.
 25. The methodaccording to claim 24 wherein the specific binding partner modulatesactivity of the PDE10 polypeptide.
 26. The method according to claim 25wherein the compound inhibits activity of the PDE10 polypeptide.
 27. Themethod according to claim 25 wherein the compound enhances activity ofthe PDE10 polypeptide.
 28. A method to identify a specific bindingpartner compound of the PDE 10 polynucleotide according to claim 5comprising the steps of: a) contacting the PDE10 polynucleotide with acompound under conditions which permit binding between the compound andthe PDE10 polynucleotide; b) detecting binding of the compound to thePDE10 polynucleotide; and c) identifying the compound as a specificbinding partner of the PDE10 polynucleotide.
 29. The method according toclaim 28 wherein the specific binding partner modulates expression of aPDE10 polypeptide encoded by the PDE10 polynucleotide.
 30. The methodaccording to claim 29 wherein the compound inhibits expression of thePDE10 polypeptide.
 31. The method according to claim 29 wherein thecompound enhances expression of the PDE10 polypeptide.
 32. A compoundidentified by the method according to claim 24 or
 28. 33. A compositioncomprising the compound according to claim 32 and a pharmaceuticallyacceptable carrier.